Vaccine for transcutaneous immunization

ABSTRACT

A vaccine delivered by transcutaneous immunization provides an effective treatment against infections by pathogens such as, for example, enterotoxigenic  Escherichia coli  (ETEC) and/or for symptoms of diarrheal disease caused thereby. For example, one, two, three, four, five or more antigens derived from ETEC and capable of inducing an antigen-specific immune response (e.g., toxins, colonization or virulence factors) and one or more optional adjuvant (e.g., whole bacterial ADP-ribosylating exotoxins, B subunits or toxoids thereof, detoxified mutants and derivatives thereof) are used to manufacture vaccines or to induce systemic and/or mucosal immunity.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. National Phase Application ofInternational Application No. PCT/USO2/04254, filed Feb. 13, 2002, whichclaims the benefit of U.S. Provisional Application No. 60/268,016, filedFeb. 13, 2001; U.S. Provisional Application No. 60/304,110, filed Jul.11, 2001; U.S. Provisional Application No. 60/310,447, filed Aug. 8,2001; and U.S. Provisional Application No. 60/310,483, filed Aug. 8,2001, all of which are herein incorporated by reference in theirentirety.

STATEMENT REGARDING FEDERAL SPONSORSHIP

The U.S. federal government has certain rights in this invention asprovided for under contracts MRMC/DAMD17-01-0085 and NIH/AI 45227-01.

FIELD OF THE INVENTION

The invention relates to vaccines and transcutaneous immunization totreat infections by pathogens such as, for example, enterotoxigenicEscherichia coli (ETEC) and/or other symptoms of diarrheal diseasecaused thereby.

BACKGROUND OF THE INVENTION

Skin, the largest human organ, plays an important part in the body'sdefense against invasion by infectious agents and contact with noxioussubstances. But this barrier function of the skin appears to haveprevented the art from appreciating that transcutaneous immunizationprovided an effective alternative to enteral, mucosal, and parenteraladministration of vaccines.

Anatomically, skin is composed of three layers: the epidermis, thedermis, and subcutaneous fat. Epidermis is composed of the basal, thespinous, the granular, and the cornified layers; the stratum corneumcomprises the cornified layer and lipid. The principal antigenpresenting cells of the skin, Langerhans cells, are reported to be inthe mid- to upper-spinous layers of the epidermis in humans. Dermiscontains primarily connective tissue. Blood and lymphatic vessels areconfined to the dermis and subcutaneous fat.

The stratum corneum, a layer of dead skin cells and lipids, hastraditionally been viewed as a barrier to the hostile world, excludingorganisms and noxious substances from the viable cells below the stratumcorneum. Stratum corneum also serves as a barrier to the loss ofmoisture from the skin: the relatively dry stratum corneum is reportedto have 5% to 15% water content while deeper epidermal and dermal layersare relatively well hydrated with 85% to 90% water content. Onlyrecently has the secondary protection provided by antigen presentingcells (e.g., Langerhans cells) been recognized. Moreover, the ability toimmunize through the skin with or without penetration enhancement (i.e.,transcutaneous immunization) using a skin-active adjuvant has only beenrecently described. Although undesirable skin reactions such as atopyand dermatitis were known in the art, recognition of the therapeuticadvantages of transcutaneous immunization (TCl) might not have beenappreciated in the past because the skin was believed to provide abarrier to the passage of molecules larger than about 500 daltons.

We have shown that a variety of adjuvants are effectively administeredby TCl to elicit systemic and regional antigen-specific immune responsesto a separate, co-administered antigen. See WO 98/20734, WO 99/43350,and WO 00/61184; U.S. Pat. Nos. 5,910,306 and 5,980,898; and U.S. patentapplication Ser. Nos. 09/257,188; 09/309,881; 09/311,720; 09/316,069;09/337,746; and 09/545,417. For example, adjuvants like ADP-ribosylatingexotoxins are safe and effective when applied epicutaneously, incontrast to the disadvantages associated with their use whenadministered by an enteral, mucosal, or parenteral route.

U.S. Pat. Nos. 4,220,584 and 4,285,931 use E. coli heat-labileenterotoxin to immunize against E. coli-induced diarrhea. Rabbits wereintramuscularly injected with the immunogen and Freund's adjuvant.Protection against challenge with toxin and neutralization of toxiceffects on ileal loop activity was shown. U.S. Pat. No. 5,182,109describes combining vaccine and toxin (e.g., E. coli heat-labile toxin)and administration in injectable, spray, or oral form. Neutralizationwas demonstrated with colostrum of immunized cows. Mutant versions ofenterotoxin have also been described to retain immunogenicity andeliminate toxicity (e.g., U.S. Pat. Nos. 4,761,372 and 5,308,835).

Novel and inventive vaccine formulations, as well as processes formaking and using them, are disclosed herein. In particular, TCl and theadvantages derived therefrom in human vaccination to treat diarrhealdisease are demonstrated. An important showing is that competition amongdifferent antigens in a multivalent vaccine was not an obstacle whenadministered by transcutaneous immunization. Other advantages of theinvention are discussed below or would be apparent from the disclosureherein.

SUMMARY OF THE INVENTION

Immunogens comprised of at least one adjuvant and/or one or moreantigens capable of inducing an immune response against pathogens likeenterotoxigenic E. coli (ETEC) are provided for immunization. Theadjuvant may be an ADP-ribosylating exotoxin (e.g., E. coli heat-labileenterotoxin, cholera toxin, diphtheria toxin, pertussis toxin) orderivatives thereof having adjuvant activity; the antigen may be derivedfrom a bacterial toxin (e.g., heat-labile or heat-stable enterotoxin) ora colonization or a virulence factor (e.g., CFA/I, CS1, CS2, CS3, CS4,CS5, CS6, CS17, PCF 0166) or peptide fragments or conjugates thereofhaving immunogenic activity. Subunit or whole-cell vaccines comprised ofan immunogen and a patch are also provided, along with methods of makingthe aforementioned products and of using them for immunization. Animmune response which is specific for molecules associated withpathogens (e.g., toxins, membrane proteins) may be induced by variousroutes (e.g., enteral, mucosal, parenteral, transcutaneous). Othertraveler's diseases of interest that can be treated includecampylobacteriosis (Campylobacter jejuni), giardiasis (Giardiaintestinalis), hepatitis (hepatitis virus A or B), malaria (Plasmodiumfalciparum, P. vivax, P. ovale, and P. malariae), shigellosis (Shigellaboydii, S. dysenteriae, S. flexneri, and S. sonnei), viralgastroenteritis (rotavirus), and combinations thereof. Effectiveness maybe assessed by clinical or laboratory criteria. Protection may beassessed using surrogate markers or directly in controlled trials.Further aspects of the invention will be apparent to a person skilled inthe art from the following detailed description and claims, andgeneralizations thereto.

DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D. Individual IgG and IgA peak fold rise in antibody titer toLT (A and B) and CS6 (C and D) among human volunteers immunized withadjuvant combined with antigen (LT+CS6), or with antigen alone (CS6).The transverse bar represents the median peak fold rise in antibodytiter.

FIG. 2A-2D. Kinetics of the anti-LT (A and B) and anti-CS6 (C and D) IgAand IgG antibody responses among volunteers immunized and boosted(arrows) using the transcutaneous route. The circles indicate thegeometric mean titer by the day after the first immunization, the barsdenote the corresponding 95% confidence intervals.

*p<0.05,**p<0.01,***p<0.001, NS non-significant, Wilcoxon signed ranktest, comparing antibody titer responses between boosting immunizations.

FIG. 3A-3D. Individual peak number of anti-LT (A and B) and anti-CS6 (Cand D) ASC per 106 PBMC among responders to the immunization withadjuvant combined with antigen (LT+CS6), by the immunization after whichthe peak value was attained.

FIG. 4A-4C. Serum IgG response to TCl with CS3 and CS6 with and withoutLTR192G adjuvant. Mice were shaved on the dorsal caudal surface at thebase of the tail 48 hr prior to vaccination. The shaved skin waspretreated by hydration with 10% glycerol and 70% isopropyl alcohol andtape stripped 10 times to disrupt the stratum corneum. Gauze patcheswere affixed to an adhesive backing and loaded with a 25 μl volume of 25μg CS6 or 25 μg CS6 with 10 μg LTR192G. The patches were applied to theprepared skin and allowed to remain in place for ˜18 hr. A group of micewas intradermally injected with a 25 μl of CS6(25 μg) at the base of thetail. All mice received a vaccination on day 0, 14 and 28. Serum sampleswere collected 14 days after the third vaccination (day 42). Panels showserum IgG titer to CS3 (A), serum IgG titer to CS6 (B), and serum IgGtiter to LTR192G (C).

FIG. 5A-5B. Serum IgG response to TCl with divalent and trivalent ETECsubunit vaccines. The vaccination site at the base of the tail wasprepared using the procedure described in FIG. 4. Gauze patches, affixedto an adhesive backing were loaded with 25 μl volume consisting of thefollowing mixtures: 25 μg CS3 and 10 μg LTR192G; 25 μg CS3, 25 μg CS6and 10 μg LTR192G. The patches were applied to the prepared skin andallowed to remain in place for ˜18 hr. All mice received atranscutaneous vaccine on day 0 and 14. Serum was collected 10 daysafter the second immunization (day 24). Panels show serum IgG titer toCS3 (A) and serum IgG titer to CS6 (B).

FIG. 6A-6B. Serum IgG response to TCl using a CS3, CS6 and LTR192Gmultivalent vaccines. The vaccination site at the base of the tail wasprepared using the procedure described in FIG. 4. Gauze patches affixedto an adhesive backing were loaded with 25 μl volume consisting of thefollowing mixtures: 25 μg CS3; 25 μg CS6; 25 μg each CS3 and CS6; 25 μgeach CS3 and CS6 and 10 μg LTR192G. The patches were applied to thepretreated skin and allowed to remain in place for ˜18 hr. All micereceived two transcutaneous vaccinations on day 0 and 14. Serum wascollected 10 days after the second immunization (day 24). Panels showserum IgG titer to CS3 (A) and serum IgG titer to CS6 (B).

FIG. 7A-7B. Lack of antibody cross-reactivity between CS3 and CS6. Thesite at the base of the tail was prepared using the procedure describedin FIG. 4. Gauze patches affixed to an adhesive backing were loaded with25 μl volume consisting of the following mixtures: 25 μg CS3 with 10 μgLTR192G (panels A and B) and 25 μg CS6 with 10 μg LTR192G (panels C andD). The patches were applied overnight (˜18 hr). All mice received twotranscutaneous vaccinations on day 0 and 14. Serum was collected 10 daysafter the second immunization (day 24). Serum IgG titers for CS3 (panelsA and C) and CS6 (panels B and D) were determined.

FIG. 8A-8C. Serum IgG subclasses elicited by transcutaneous vaccinationwith CS3 with and without LTR192G. Mice were shaved on the dorsal caudalsurface at the base of the tail 48 hr prior to vaccination. The shavedskin was pretreated by hydration with 10% glycerol and 70% isopropylalcohol. The hydrated skin was then mildly abraded with emery paper 10times. Gauze patches were affixed to an adhesive backing and loaded with25 μl of the following mixtures: 25

μg CS3 or 25 μg CS3 with or without 10 μg LTR192G. The patches wereapplied overnight (˜18 hr). All mice received three transcutaneousvaccinations on day 0, 14 and 28. Serum samples were collected 30 daysafter the third vaccination (day 58). Panels show total serum IgG titersto CS3 (A), serum IgG1 subclass to CS3 (B), and serum IgG2a subclass toCS3 (C).

FIG. 9A-9C. Serum IgG subclasses elicited by TCl with CS6 with andwithout and LTR192G. Mice were shaved on the dorsal caudal surface atthe base of the tail 48 hr prior to vaccination. The shaved skin waspretreated by hydration with 10% glycerol and 70% isopropyl alcohol. Thehydrated skin was then mildly abraded with emery paper 10 times. Gauzepatches were affixed to an adhesive backing and loaded with 25 μl of thefollowing mixtures: 25 μg CS6 or 25 μg CS3 with or without 10 μgLTR192G. The patches were applied overnight (˜18 hr). All mice receivedthree transcutaneous vaccinations on day 0, 14, and 28. Serum sampleswere collected 30 days after the third vaccination (day 58). Panels showtotal serum IgG titers to CS6 (A), serum IgG1 subclass to CS6 (B), andserum IgG2a subclass to CS6 (C).

FIG. 10A-10C. Serum IgG subclasses elicited by TCl with LTR192G. Micewere shaved on the dorsal caudal surface at the base of the tail 48 hrprior to vaccination. The shaved skin was pretreated by hydration with10% glycerol and 70% isopropyl alcohol. The hydrated skin was thenmildly abraded with emery paper 10 times. Gauze patches were affixed toan adhesive backing and loaded with 25 μl of 10 μg LTR192G. The patcheswere applied overnight (˜18 hr). All mice received three transcutaneousvaccinations on day 0, 14 and 28. Serum samples were collected 30 daysafter the third vaccination (day 58). Panels shown total serum IgGtiters to LTR192G (A), serum IgG1 subclass to LTR192G (B), and serumIgG2a subclass to LTR192G (C).

FIG. 11A-11C. Serum IgG subclasses elicited by LTR192G co-administeredwith CS3 or CS6. Mice were shaved on the dorsal caudal surface at thebase of the tail 48 hr prior to vaccination. The shaved skin waspretreated by hydration with 10% glycerol and 70% isopropyl alcohol. Thehydrated skin was then mildly abraded with emery paper 10 times. Gauzepatches were affixed to an adhesive backing and loaded with 25 μl of thefollowing: 25 μg CS3 with 10 μg LTR192G; and 25 μg CS6 with 10 μgLTR192G. The patches were applied overnight (˜18 hr). All mice receivedthree transcutaneous vaccinations on day 0, 14 and 28. Serum sampleswere collected 30 days after the third vaccination (day 58). Panels showtotal serum IgG titers to LTR192G (A), serum IgG1 subclass to LTR192G(B), and serum IgG2a subclass to LTR192G (C).

FIG. 12A-12H. Detection of CS3 specific fecal IgA (upper panels) and IgG(lower panels) following TCl. Mice were shaved on the dorsal caudalsurface at the base of the tail 48 hr prior to vaccination. The shavedskin was pretreated by hydration with 10% glycerol and 70% isopropylalcohol. The hydrated skin was then tape stripped 10 times. Gauzepatches were affixed to an adhesive backing and loaded with 25 μl of thefollowing mixtures: phosphate buffered saline (panels A and E); 25 μgCS3 (panels B and F); and 25 μg CS3 with 10 μg LTR192G (panels C and G).The patches were applied overnight (˜18 hr). A group of mice wasvaccinated by intradermal (ID) injection of 25 μg CS3 (panels D and H).All mice received three vaccinations on day 0, 14 and 28. Fecal sampleswere collected one week after the third immunization (day 35). Thesamples were processed and evaluated for fecal IgA (panels A-D) and IgG(panels E-H) against CS3.

FIG. 13A-13H. Detection of CS6 specific fecal IgA (upper panels) and IgG(lower panels) following TCl. Mice were shaved on the dorsal caudalsurface at the base of the tail 48 hr prior to vaccination. The shavedskin was pretreated by hydration with 10% glycerol and 70% isopropylalcohol. The hydrated skin was then tape stripped 10 times. Gauzepatches were affixed to an adhesive backing and loaded with 25 μl of thefollowing mixtures: phosphate buffered saline (panels A and E); 25 μgCS6 (panels B and F); and 25 μg CS6 with 10 μg LTR192G (panels C and G).The patches were applied overnight (˜18 hr). A group of mice wasvaccinated by intradermal (ID) injection of 25 μg CS6 (panels D and H).All mice received three vaccinations on day 0, 14 and 28. Fecal sampleswere collected one week after the third immunization (day 35). Thesamples were processed and evaluated for fecal IgA (panels A-D) and IgG(panels E-H) against CS6.

FIG. 14A-14H. Detection of LTR192G specific fecal IgA (upper panels) andIgG (lower panels) following TCl. Mice were shaved on the dorsal caudalsurface at the base of the tail 48 hr prior to vaccination. The shavedskin was pretreated by hydration with 10% glycerol and 70% isopropylalcohol. The hydrated skin was then tape stripped 10 times. Gauzepatches were affixed to an adhesive backing and loaded with 25 μl of thefollowing mixtures: phosphate buffered saline (panels A and E); 10 μgLTR192G (panels B and F); and 25 μg CS3 with 10 μg LTR192G (panels C andG); and 25 μg CS6 and 10 μg LTR192G (panel D and H). The patches wereapplied overnight (˜18 hr). All mice received three vaccinations on day0, 14 and 28. Fecal samples were collected one week after the thirdimmunization (day 35). The samples were processed and evaluated forfecal IgA (panels A-D) and IgG (panels E-H) against LTR192G.

FIG. 15A-15D. Detection of CS3, CS6 and LTR192G specific antibodysecreting cells (ASC) in the spleen of mice transcutaneously vaccinatedwith monovalent and divalent ETEC subunit vaccines. Mice were shaved onthe dorsal caudal surface at the base of the tail 48 hr prior tovaccination. The shaved skin was pretreated by hydration with 10%glycerol and 70% isopropyl alcohol. The hydrated skin was then tapestripped 10 times. Gauze patches were affixed to an adhesive backing andloaded with 25 μl of the following mixtures: phosphate buffered saline(vehicle); 25 μg CS3; 25 μg CS6; 25 μg CS3 with 10 μg LTR192G; and 25 μgCS6 with 10 μg LTR192G. The patches were applied overnight (˜18 hr). Inaddition, groups of mice were vaccinated by intradermal (ID) injectionat the base of the tail with 25 μg of CS3 or CS6. All mice werevaccinated three times on day 0, 14 and 28. The spleen was harvested 30days after the third immunization (day 58). Panels show CS3-specificIgA-ASC (A) and IgG-ASC (B); CS6-specific IgA-ASC (C) and IgG-ASC (D),and LTR192G-specific IgA-ASC (A and C) and IgG-ASC (B and D).

FIG. 16A-16B. Detection of CS3, CS6 and LTR192G specific antibodysecreting cells (ASC) in the spleen of mice transcutaneously vaccinatedwith trivalent ETEC subunit vaccine. Mice were shaved on the dorsalcaudal surface at the base of the tail 48 hr prior to vaccination. Theshaved skin was pretreated by hydration with 10% glycerol and 70%isopropyl alcohol. The hydrated skin was then tape stripped 10 times.Gauze patches were affixed to an adhesive backing and loaded with 25 μlof a mixture of the following formulation: phosphate buffered saline(vehicle); 25 μg CS3; 25 μg CS6; 25 μg CS3 with 10 μg LTR192G; 25 μgCS3/25 g CS6/10 μg LTR192G. The patches were applied overnight (˜18 hr).Mice were vaccinated three times on day 0, 14 and 28. The spleen washarvested 30 days after the third immunization (day 58). Panels showIgA-ASC specific for CS3, CS6 and LTR192G (A) and IgG-ASC specific forCS3, CS6 and LTR192G (B).

FIG. 17A-17B. Detection of CS3, CS6 and LTR192G specific antibodysecreting cells (ASC) in the inguinal lymph nodes of micetranscutaneously vaccinated with monovalent and divalent ETEC subunitvaccines. Mice were shaved on the dorsal caudal surface at the base ofthe tail 48 hr prior to vaccination. The shaved skin was pretreated byhydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skinwas then tape stripped 10 times. Gauze patches were affixed to anadhesive backing and loaded with 25 μl of the following mixtures:phosphate buffered saline (vehicle); 25 μg CS3; 25 μg CS6; 25 μg CS3with 10 μg LTR192G; and 25 μg CS6 with 10 μg LTR192G. The patches wereapplied overnight (˜18 hr). In addition, separate groups of mice werevaccinated by intradermal (ID) injection at the base of the tail with 25μg of CS3 or CS6. All mice were vaccinated three times on day 0, 14 and28. Inguinal lymph nodes were collected 30 days after the thirdimmunization (day 58). Panels show CS3-specific IgG-ASC (A),CS6-specific IgG-ASC (B), and LTR192G-specific IgG-ASC (A and B).

FIG. 18. Detection of CS3, CS6 and LTR192G specific antibody secretingcells (IgG-ASC) in the inguinal lymph nodes of mice transcutaneouslyvaccinated with trivalent ETEC subunit vaccine. Mice were shaved on thedorsal caudal surface at the base of the tail 48 hr prior tovaccination. The shaved skin was pretreated by hydration with 10%glycerol and 70% isopropyl alcohol. The hydrated skin was then tapestripped 10 times. Gauze patches were affixed to an adhesive backing andloaded with 25 μl of a mixture consisting of 25 μg CS3, 25 μg CS6 and 10μg LTR192G. The patches were applied overnight (˜18 hr). All mice werevaccinated three times on day 0, 14 and 28. Inguinal lymph nodes werecollected 30 days after the third immunization (day 58).

FIG. 19A-19B. Serum IgG response to TCl with CFA/I with and withoutLTR192G adjuvant. Mice were shaved on the dorsal caudal surface at thebase of the tail 48 hr prior to vaccination. The shaved skin waspretreated by hydration with 10% glycerol and 70% isopropyl alcohol andmildly abraded with emery paper 5 times to disrupt the stratum corneum.Gauze patches were affixed to an adhesive backing and loaded with a 25μl volume of 25 μg CFA/I and 25 μg CFA/I with 10 μg LTR192G. The patcheswere applied to the prepared skin and allowed to remain in place for ˜18hr. Separate groups of mice was intradermally injected with a 25 μl ofCFA/I (25 μg) at the base of the tail. All mice received a vaccinationon day 0 and 14. Serum samples were collected 10 days after the secondvaccination (day 24). Panels show serum IgG titer to CFA/I (A) and serumIgG titer to LTR192G (B).

FIG. 20A-20H. Detection of CFA/I specific fecal IgA (upper panels) andIgG (lower panels) following TCl. Mice were shaved on the dorsal caudalsurface at the base of the tail 48 hr prior to vaccination. The shavedskin was pretreated by hydration with 10% glycerol and 70% isopropylalcohol. The hydrated skin was then mildly abraded with emery paper 5times. Gauze patches were affixed to an adhesive backing and loaded with25 μl volume of 25 μg of the following mixtures: phosphate bufferedsaline (panels A and E); 25 of the following mixtures: phosphatebuffered saline (Panels A and E); 25 μg CFA/I (panels B and F); and 25μg CFA/I with 10 μg LTR192G (panels C and G). The patches were appliedovernight (˜18 hr). A group of mice was vaccinated by intradermal (D)injection of 25 μg CFA/I (panels D and H). All mice received threevaccinations on day 0, 14 and 28. Fecal samples were collected two weeksafter the third immunization (day 42). The samples were processed andevaluated for fecal IgA (panels A-D) and IgG (panels E-H) against CFA/I.

FIG. 21. Serum IgG response to TCl with a tetravalent ETEC subunitvaccine. Mice were shaved on the dorsal caudal surface at the base ofthe tail 48 hr prior to vaccination. The shaved skin was pretreated byhydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skinwas mildly abraded with emery paper 5 times. Gauze patches, affixed toan adhesive backing were loaded with a mixture consisting of 25 μgCFA/I, 25 μg CS3, 25 μg CS6 and 10 μg LTR192G. The patches were appliedto the prepared skin and allowed to remain in place for ˜18 hr. All micereceived a transcutaneous vaccination on day 0 and 14. Serum wascollected 10 days after the second immunization (day 24).

FIG. 22A-22F. Detection of fecal IgA (upper panels) and IgG (lowerpanels) antibodies to colonization factor antigens following TCl withthe tetravalent ETEC vaccine. Mice were shaved on the dorsal caudalsurface at the base of the tail 48 hr prior to vaccination. The shavedskin was pretreated by hydration with 10% glycerol and 70% isopropylalcohol. The hydrated skin was then mildly abraded with emery paper.Gauze patches were affixed to an adhesive backing and loaded with thetetravalent vaccine: 25 μg CFA/I, 25 μg CS, 25 μg CS6 and 10 μg LTR192G.The patches were applied overnight (˜18 hr). All mice were vaccinated byintradermal (D) injections of 25 μg CFA/I (panels D and H). All micereceived three vaccinations on day 0, 14 and 28. Fecal samples werecollected two weeks after the third immunization (day 42). The sampleswere processed and evaluated for fecal IgA to CFA/I(A), CS3 (B), and CS6(C). Processed samples were also evaluated for fecal IgG to CFA/I (D),CS3 (E), and CS6 (F).

FIG. 23A-23F. Detection of fecal IgA (upper panels) and IgG (lowerpanels) antibodies to LTR192G following TCl with the tetravalent ETECvaccine. Mice were shaved on the dorsal caudal surface at the base ofthe tail 48 hr prior to vaccination. The shaved skin was pretreated byhydration and abrasion as described in FIG. 18. Gauze patches wereaffixed to an adhesive backing and loaded with the following: 10 gLTR192G (mLT); 25 μg CFA/I and LTR192G; or 25 μg CFA/I, 25 μg CS, 25 μgCS6 and 10 μg LTR192G. The patches were applied overnight (˜18 hr). Allmice received three vaccinations on day 0,14 and 28. Fecal samples werecollected one week after the third immunization (day 35). Samples wereprocessed and evaluated for fecal IgA to LTR192G (panels A-c) and forfecal IgG to LTR192G (panels D-F).

FIG. 24. Transcutaneous vaccination with CS3 and LTR192G subunitvaccines elicit serum antibodies that recognize CS3 expressing ETECwhole cells. Mice were shaved at the base of the tail by standardprocedures. The shaved skin was tape stripped 10 times immediately priorto application of the patch. A gauze patch affixed to an adhesivebacking was loaded with 25 μg CS3 and 10 μg LTR192G immediately prior toapplication. The patch was applied for ˜18 hr. A group of 10 micereceived two patches on day 0 and day 14. Serum was collected 10 daysafter the second immunization (day 24). The serum was evaluated forantibodies to CS3, LTR192G and ETEC whole cells (E243778).

FIG. 25. Transcutaneous vaccination with killed enterotoxigenic E. coliwhole cells (EWC). EWC were prepared by culturing ETEC (strain E243778)in bacterial broth. The cells were harvested by centrifugation andinactivated by overnight (room temperature) fixation with 2.5% formalin.The inactivated, killed whole cells were washed with phosphate bufferedsaline to remove the formalin. Prior to immunization, the mice wereshaved at the base of the tail. The shaved skin was tape stripped 10times immediately prior to application of the patch. The gauze patch onan adhesive backing was loaded with 10⁹ EWC's and 10 μg LTR192G. A groupof 10 mice received were transcutaneously vaccinated on day 0 and 14.Serum was collected 10 days after the second immunization. Sera wereevaluated for antibodies to EWC and LTR192G using the ELISA method asdescribed in Materials and Methods. The results in FIG. 22 show thattranscutaneous vaccination with killed bacterial whole cells did elicitantibodies that recognized whole cells and LTR192G adjuvant. Theseresults demonstrate that killed ETEC bacteria can be applied to skinwith the adjuavant and elicit specific immunity. These results aresignificant in that this is the first demonstration that TCl isapplicable for subunit vaccines and for delivery of killed whole cellvaccines.

FIG. 26A-26B. Transcutaneous vaccination with a multivalent ETEC vaccineconsisting of multiple colonization factors and two enterotoxins, LT andST. Mice were shaved on the dorsal caudal surface at the base of thetail 48 hr prior to vaccination. The shaved skin was pretreated byhydration with 10% glycerol and 70% isopropyl alcohol and tape stripped10 times to disrupt the stratum comeum. Gauze patches were affixed to anadhesive backing and loaded with a 25 μl volume of 25 μg CS3/25 μg CS6;26 μg CS3/25 μg CS6/10 μg LTR192G and 25 μg CS3/25 μg CS6/10 μgLTR192G/8 μg STa. The patches were applied to the prepared skin andallowed to remain in place for ˜m18 hr. All mice received a vaccinationon day 0, 14 and 28. Serum samples were collected 14 days after thesecond vaccination (day 42). Panels show serum IgG titer to CS3 (A) andserum IgG titer to CS6 (B).

FIG. 27. Wet and dry patch formulations are suitable for manufacturingarticles for TCl. In these studies LT was used as an example forpreparing different liquid and patch formulations. Briefly, LT wasformulated in phosphate buffered saline and 5% lactose; LT was blendedwith an adhesive (KLUCEL) and spread as a thin film over an occlusivebacking and allowed to air-dry at room temperature; LT solution wasdirectly applied to a gauze patch surface and air-dried prior to use;and LT solution was applied to a gauze patch and administered as a fullyhydrated patch. For mice receiving the liquid LT formulation, 10 μg LTwas applied directly to the skin for 1 hr (with or without covering withgauze) and rinsed off. For mice receiving patches, the different patchformulations were applied for ˜24 hr before removal. The skin washydrated with 10% glycerol and 70% isopropyl alcohol followed by mildlydisrupting the stratum comeum with a pumice-containing swab(PDI/NicePak). All mice received two vaccinations on day 0 and day 14with an equivalent of 10 μg (˜1 cm² area). Serum was collected two weekslater (day 28) and evaluated for serum antibodies to LT. Aqueoussolutions, protein-in-adhesive, air-dried and fully hydrated patchformulations are suitable for transcutaneous delivery of ETEC antigens.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

A system for transcutaneous immunization (TCl) is provided which inducesan immune response (e.g., humoral and/or cellular effector specific foran antigen) in an animal or human. The delivery system provides simple,epicutaneous application of a formulation comprised of one or moreadjuvants, antigens, and/or polynucleotides (encoding adjuvant and/orantigen) to the skin of an animal or human subject. An antigen-specificimmune response is thereby elicited against one or more pathogens likeenterotoxigenic E. coli (ETEC) with or without the aid of chemicaland/or physical penetration enhancement. At least one ingredient orcomponent of the formulation (i.e., antigen or adjuvant) may be providedin dry form prior to administration of the formulation and/or as part ofa patch. This system may also be used in conjunction with conventionalenteral, mucosal, or parenteral immunization techniques.

Activation of one or more of adjuvant, antigen, and antigen presentingcell (APC) may assist in the promoting the immune response. The APCprocesses the antigen and then presents one or more epitopes to alymphocyte. Activation may promote contact between the formulation andthe APC (e.g., Langerhans cells, other dendritic cells, macrophages, Blymphocytes), uptake of the formulation by the APC, processing ofantigen and/or presentation of epitopes by the APC, migration and/ordifferentiation of the APC, interaction between the APC and thelymphocyte, or combinations thereof. The adjuvant by itself may activatethe APC. For example, a chemokine may recruit and/or activate antigenpresenting cells to a site. In particular, the antigen presenting cellmay migrate from the skin to the lymph nodes, and then present antigento a lymphocyte, thereby inducing an antigen-specific immune response.Furthermore, the formulation may directly contact a lymphocyte whichrecognizes antigen, thereby inducing an antigen-specific immuneresponse.

In addition to eliciting immune reactions leading to activation and/orexpansion of antigen-specific B-cell and/or T-cell populations,including antibodies and cytotoxic T lymphocytes (CTL), the inventionmay positively and/or negatively regulate one or more components of theimmune system by using transcutaneous immunization to affectantigen-specific helper (Th1 and/or Th2) or delayed-typehypersensitivity T-cell subsets (T_(DTH)). This can be exemplified bythe differential behavior of cholera toxin and E. coli heat-labileenterotoxin which can result in different T-helper responses. Thedesired immune response induced by the invention is preferably systemicor regional (e.g., mucosal) but is usually not undesirable immuneresponses (e.g., atopy, dermatitis, eczema, psoriasis, or other allergicor hypersensitivity reactions). As seen herein, the immune responseselicited are of the quantity and quality that provide therapeutic orprophylactic immune responses useful treatment of infectious disease.

TCl may be practiced with or without skin penetration. For example,chemical or physical penetration enhancement techniques may be used aslong as the skin is not perforated through the dermal layer. Hydrationof the intact or skin before, during, or immediately after applicationof the formulation is preferred and may be required in some or manyinstances. For example, hydration may increase the water content of thetopmost layer of skin (e.g., stratum corneum or superficial epidermislayer exposed by penetration enhancement techniques) above 25%, 50% or75%.

Skin may be swabbed with an applicator (e.g., adsorbent material on apad or stick) containing hydration or chemical penetration agents orthey may be applied directly to skin. For example, aqueous solutions(e.g., water, saline, other buffers), acetone, alcohols (e.g., isopropylalcohol), detergents (e.g., sodium dodecyl sulfate), depilatory orkeratinolytic agents (e.g., calcium hydroxide, salicylic acid, ureas),humectants (e.g., glycerol, other glycols), polymers (e.g., polyethyleneor propylene glycol, polyvinyl pyrrolidone), or combinations thereof maybe used or incorporated in the formulation. Similarly, abrading the skin(e.g., abrasives like an emery board or paper, sand paper, fibrous pad,pumice), removing a superficial layer of skin (e.g., peeling orstripping with an adhesive tape), microporating the skin using an energysource (e.g., heat, light, sound, electrical, magnetic) or a barrierdisruption device (e.g., gun, microneedle), or combinations thereof mayact as a physical penetration enhancer. See WO98/29134 for microporationof skin and U.S. Pat. No. 6,090,790 for microneedles and U.S. Pat. No.6,168,587 for transdermal guns which might be adapted for use intranscutaneous vaccination. The objective of chemical or physicalpenetration enhancement in conjunction with TCl is to remove at leastthe stratum corneum or deeper epidermal layer without perforating theskin through to the dermal layer. This is preferably accomplished withminor discomfort at most to the human or animal subject and withoutbleeding at the site. For example, applying the formulation to intactskin may not involve thermal, optical, sonic, or electromagnetic energyto perforate layers of the skin below the stratum corneum or epidermis.

Formulations which are useful for vaccination are also provided as wellas processes for their manufacture. The formulation may be in dry orliquid form. A dry formulation is more easily stored and transportedthan conventional vaccines, it breaks the cold chain required from thevaccine's place of manufacture to the locale where vaccination occurs.Without being limited to any particular mode of action, another way inwhich a dry formulation may be an improvement over liquid formulationsis that high concentrations of a dry active component of the formulation(e.g., one or more adjuvants and/or antigens) may be achieved bysolubilization directly at the site of immunization over a short timespan. Moisture from the skin (e.g., perspiration) and an occlusivedressing may hasten this process. In this way, it is possible that aconcentration approaching the solubility limit of the active ingredientmay be achieved in situ. Alternatively, the dry, active ingredient ofthe formulation per se may be an improvement by providing a solidparticulate form that is taken up and processed by antigen presentingcells. These possible mechanisms are discussed not to limit the scope ofthe invention or its equivalents, but to provide insight into theoperation of the invention and to guide the use of this formulation inimmunization and vaccination.

The formulation may be provided as a liquid: cream, emulsion, gel,lotion, ointment, paste, solution, suspension, or other liquid forms.Dry formulations may be provided in various forms: for example, fine orgranulated powders, uniform films, pellets, and tablets. The formulationmay be dissolved and then dried in a container or on a flat surface(e.g., skin), or it may simply be dusted on the flat surface. It may beair dried, dried with elevated temperature, freeze or spray dried,coated or sprayed on a solid substrate and then dried, dusted on a solidsubstrate, quickly frozen and then slowly dried under vacuum, orcombinations thereof. If different molecules are active ingredients ofthe formulation, they may be mixed in solution and then dried, or mixedin dry form only. Compartments or chambers of the patch may be used toseparate active ingredients so that only one of the antigens oradjuvants is kept in dry form prior to administration; separating liquidand solid in this manner allows control over the time and rate of thedissolving of at least one dry, active ingredient.

A “patch” refers to a product which includes a solid substrate (e.g.,occlusive or non-occlusive surgical dressing) as well as at least oneactive ingredient. Liquid may be incorporated in a patch (i.e., a wetpatch). One or more active components of the formulation may be appliedon the substrate, incorporated in the substrate or adhesive of thepatch, or combinations thereof. A dry patch may or may not use a liquidreservoir to solubilize the formulation.

Formulation in liquid or solid form may be applied with one or moreadjuvants and/or antigens both at the same or separate sites orsimultaneously or in frequent, repeated applications. The patch mayinclude a controlled-release reservoir or a rate-controlling matrix ormembrane may be used which allows stepped release of adjuvant and/orantigen. It may contain a single reservoir with adjuvant and/or antigen,or multiple reservoirs to separate individual antigens and adjuvants.The patch may include additional antigens such that application of thepatch induces an immune response to multiple antigens. In such a case,antigens may or may not be derived from the same source, but they willhave different chemical structures so as to induce an immune responsespecific for different antigens. Multiple patches may be appliedsimultaneously; a single patch may contain multiple reservoirs. Foreffective treatment, multiple patches may be applied at intervals orconstantly over a period of time; they may be applied at differenttimes, for overlapping periods, or simultaneously. At least one adjuvantand/or adjuvant may be maintained in dry form prior to administration.Subsequent release of liquid from a reservoir or entry of liquid into areservoir containing the dry ingredient of the formulation will at leastpartially dissolve that ingredient.

Solids (e.g., particles of nanometer or micrometer dimensions) may alsobe incorporated in the formulation. Solid forms (e.g., nanoparticles ormicroparticles) may aid in dispersion or solubilization of activeingredients; assist in carrying the formulation through superficiallayers of the skin; provide a point of attachment for adjuvant, antigen,or both to a substrate that can be opsonized by antigen presentingcells, or combinations thereof. Prolonged release of the formulationfrom a porous solid formed as a sheet, rod, or bead acts as a depot.

The formulation may be manufactured under aseptic conditions acceptableto appropriate regulatory agencies (e.g., Food and Drug Administration)for biologicals and vaccines. Optionally, components such as dessicants,excipients, stabilizers, humectants, preservatives, adhesives, patchmaterials, or combinations thereof may be included in the formulationeven though they are immunologically inactive. They may, however, haveother desirable properties or characteristics.

A single or unit dose of formulation suitable for administration isprovided. The amount of adjuvant or antigen in the unit dose may beanywhere in a broad range from about 0.001 μg to about 10 mg. This rangemay be from about 0.1 μg to about 1 mg; a narrower range is from about 5μg to about 500 μg. Other suitable ranges are between about 1 μg andabout 10 μg, between about 10 μg and about 50 μg, between about 50 μgand about 200 μg, and between about 1 mg and about 5 mg. A preferreddose for a toxin is about 50 μg or 100 μg or less (e.g., from about 1 μgto about 50 μg or 100 μg). The ratio between antigen and adjuvant may beabout 1:1 (e.g., E. coli heat-labile enterotoxin when it is both antigenand adjuvant) but higher ratios may be suitable for poor antigens (e.g.,about 1:10 or less), or lower ratios of antigen to adjuvant may also beused (e.g., about 10:1 or more). The native ratios between LT and ETECantigens may be used for whole-cell or lysate formulations.

A formulation comprising adjuvant and antigen or polynucleotide may beapplied to skin of a human or animal subject, antigen is presented toimmune cells, and an antigen-specific immune response is induced. Thismay occur before, during, or after infection by pathogen. Only antigenor polynucleotide encoding antigen may be required, but no additionaladjuvant, if the immunogenicity of the formulation is sufficient to notrequire adjuvant activity. The formulation may include an additionalantigen such that application of the formulation induces an immuneresponse against multiple antigens (i.e., multivalent). In such a case,antigens may or may not be derived from the same source, but theantigens will have different chemical structures so as to induce immuneresponses specific for the different antigens. Antigen-specificlymphocytes may participate in the immune response and, in the case ofparticipation by B lymphocytes, antigen-specific antibodies may be partof the immune response. The formulations described above may includedessicants, excipients, humectants, stabilizers, preservatives,adhesives, and patch materials known in the art.

The invention is used to treat a subject (e.g., a human or animal inneed of treatment such as prevention of disease, protection from effectsof infection, therapy of existing disease or symptoms, or combinationsthereof). When the antigen is derived from a pathogen, the treatment mayvaccinate the subject against infection by the pathogen or against itspathogenic effects such as those caused by toxin secretion. Theinvention may be used therapeutically to treat existing disease,protectively to prevent disease, to reduce the severity and/or durationof disease, to ameliorate symptoms of disease, or combinations thereof.

The application site may be protected with anti-inflammatorycorticosteroids such as hydrocortisone, triamcinolone and mometazone ornon-steroidal anti-inflammatory drugs (NSAID) to reduce possible localskin reaction or modulate the type of immune response. Similarly,anti-inflammatory steroids or NSAID may be included in the patchmaterial, or liquid or solid formulations; and corticosteroids or NSAIDmay be applied after immunization. IL-10, TNF-α, other immunomodulatorsmay be used instead of the anti-inflammatory agents. Moreover, theformulation may be applied to skin overlying more than one draininglymph node field using either single or multiple applications. Theformulation may include additional antigens such that applicationinduces an immune response to multiple antigens. In such a case, theantigens may or may not be derived from the same source, but theantigens will have different chemical structures so as to induce animmune response specific for the different antigens. Multi-chamberedpatches could allow more effective delivery of multivalent vaccines aseach chamber covers different antigen presenting cells. Thus, antigenpresenting cells would encounter only one antigen (with or withoutadjuvant) and thus would eliminate antigenic competition and therebyenhancing the response to each individual antigen in the multivalentvaccine.

The formulation may be epicutaneously applied to skin to prime or boostthe immune response in conjunction with penetration techniques or otherroutes of immunization. Priming by transcutaneous immunization (TCl)with either single or multiple applications may be followed withenteral, mucosal, parenteral, and/or transdermal techniques for boostingimmunization with the same or altered antigens. Priming by enteral,mucosal, parenteral, and/or transdermal immunization with either singleor multiple applications may be followed with transcutaneous techniquesfor boosting immunization with the same or altered antigens. It shouldbe noted that TCl is distinguished from conventional topical techniqueslike mucosal or transdermal immunization because the former requires amucous membrane (e.g., lung, mouth, nose, rectum) not found in the skinand the latter requires perforation of the skin through the dermis. Theformulation may include additional antigens such that application toskin induces an immune response to multiple antigens.

In addition to antigen and adjuvant, the formulation may comprise avehicle. For example, the formulation may comprise an AQUAPHOR, FREUND,RIBI or SYNTEX emulsion; water-in-oil emulsions (e.g., aqueous creams,ISA-720), oil-in-water emulsions (e.g., oily creams, ISA-51, MF59),microemulsions, anhydrous lipids and oil-in-water emulsions, other typesof emulsions; gels, fats, waxes, oil, silicones, and humectants (e.g.,glycerol).

Antigen may be derived from any pathogen that infects a human or animalsubject (e.g., bacterium, virus, fungus, or protozoan). The chemicalstructure of the antigen may be described as one or more ofcarbohydrate, fatty acid, and protein (e.g., glycolipid, glycoprotein,lipoprotein). Proteinaceous antigen is preferred. The molecular weightof the antigen may be greater than 500 daltons, 800 daltons, 1000daltons, 10 kilodaltons, 100 kilodaltons, or 1000 kilodaltons. Chemicalor physical penetration enhancement may be preferred for macromolecularstructures like cells, viral particles, and molecules of greater thanone megadalton (e.g., CS6 antigen), but techniques like hydration andswabbing with a solvent may be sufficient to induce immunization acrossthe skin. Antigen may be obtained by recombinant techniques, chemicalsynthesis, or at least partial purification from a natural source. Itmay be a chemical or recombinant conjugates: for example, linkagebetween chemically reactive groups or protein fusion. Antigen may beprovided as a live cell or virus, an attenuated live cell or virus, akilled cell, or an inactivated virus. Alternatively, antigen may be atleast partially purified in cell-free form (e.g., cell or viral lysate,membrane or other subcellular fraction). Because most adjuvants wouldalso have immunogenic activity and would be considered antigens,adjuvants would also be expected to have the aforementioned propertiesand characteristics of antigens.

The choice of adjuvant may allow potentiation or modulation of theimmune response. Moreover, selection of a suitable adjuvant may resultin the preferential induction of a humoral or cellular immune response,specific antibody isotypes (e.g., IgM, IgD, IgA1, IgA2, IgE, IgG1, IgG2,IgG3, and/or IgG4), and/or specific T-cell subsets (e.g., CTL, Th1, Th2and/or T_(DTH)). The adjuvant is preferably a chemically activated(e.g., proteolytically digested) or genetically activated (e.g.,fusions, deletion or point mutants) ADP-ribosylating exotoxin or Bsubunit thereof. Adjuvant, antigen, or both may optionally be providedin the formulation with a polynucleotide (e.g., DNA, RNA, cDNA, cRNA)encoding the adjuvant or antigen as appropriate. Covalently closed,circular DNA such as plasmids are preferred forms of the polynucleotide;however, linear forms may also be used. The polynucleotide may include aregion such as an origin of replication, centromere, telomere, promoter,enhancer, silencer, transcriptional initiation or termination signal,splice acceptor or donor site, ribosome binding site, translationalinitiation or termination signal, polyadenylation signal, cellularlocalization signal, protease cleavage site, polylinker site, orcombinations thereof as are found in expression vectors.

An “antigen” is an active component of the formulation which isspecifically recognized by the immune system of a human or animalsubject after immunization or vaccination. The antigen may comprise asingle or multiple immunogenic epitopes recognized by a B-cell receptor(i.e., secreted or membrane-bound antibody) or a T-cell receptor.Proteinaceous epitopes recognized by T-cell receptors have typicallengths and conserved amino acid residues depending on whether they arebound by major histocompatibility complex (MHC) Class I or Class IImolecules on the antigen presenting cell. In contrast, proteinaceousepitopes recognized antibody may be of variable length including short,extended oligopeptides and longer, folded polypeptides. Single aminoacid differences between epitopes may be distinguished. The antigen iscapable of inducing an immune response against a molecule of a pathogen(e.g., a CS6 antigen is capable of inducing a specific immune responseagainst the CS6 molecule of ETEC). Thus, antigen is usually identical orat least derived from the chemical structure of a specific molecule ofthe pathogen, but mimetics which are only distantly related to suchchemical structures may also be successfully used.

An “adjuvant” is an active component of the formulation to assist ininducing an immune response to the antigen. Adjuvant activity is theability to increase the immune response to a heterologous antigen (i.e.,antigen which is a separate chemical structure from the adjuvant) byinclusion of the adjuvant itself in a formulation or in combination withother components of the formulation or particular immunizationtechniques. As noted above, a molecule may contain both antigen andadjuvant activities by chemically conjugating antigen and adjuvant orgenetically fusing coding regions of antigen and adjuvant; thus, theformulation may contain only one ingredient or component.

The term “effective amount” is meant to describe that amount of adjuvantor antigen which induces an antigen-specific immune response. A“subunit” immunogen or vaccine is a formulation comprised of activecomponents (e.g., adjuvant, antigen) which have been isolated from othercellular or viral components of the pathogen (e.g., membrane orpolysaccharide components like endotoxin) by recombinant techniques,chemical synthesis, or at least partial purification from a naturalsource.

Induction of an immune response may provide a treatment such as, forexample, prophylactic or therapeutic vaccination for an infectiousdisease. A product or method “induces” when its presence or absencecauses a statistically significant change in the immune response'smagnitude and/or kinetics; change in the induced elements of the immunesystem (e.g., humoral vs. cellular, Th1 vs. Th2); effect on the healthand well-being of the subject; or combinations thereof.

The term “draining lymph node field” as used in the invention means ananatomic area over which the lymph collected is filtered through a setof defined lymph nodes (e.g., cervical, axillary, inguinal,epitrochelear, popliteal, those of the abdomen and thorax). Thus, thesame draining lymph node field may be targeted by immunization (e.g.,enteral, mucosal, parenteral, transcutaneous, transdermal) within thefew days required for antigen presenting cells to migrate to the lymphnodes if the sites and times of immunization are spaced to bringdifferent components of the formulation together (e.g., two closelyspaced patches with either adjuvant or antigen may be effective whenneither alone could successfully used). For example, a patch deliveringadjuvant by the transcutaneous technique may be placed on the same armas is injected with a conventional vaccine to boost its effectiveness inelderly, pediatric, or other immunologically compromised populations. Incontrast, applying patches to different limbs may prevent anadjuvant-containing patch from boosting the effectiveness of a patchcontaining only antigen.

Without being bound to any particular theory for the operation of theinvention but only to provide an explanation for our observations, wehypothesize that this transcutaneous delivery system carries antigen tocells of the immune system where an immune response is induced. Theantigen may pass through the normally present protective outer layers ofthe skin (i.e., stratum corneum) and induce the immune responsedirectly, or through an antigen presenting cell population in theepidermis (e.g., macrophage, tissue macrophage, Langerhans cell, otherdendritic cells, B lymphocyte, or Kupffer cell) that presents processedantigen to lymphocytes. Thus, with or without penetration enhancementtechniques, the dermis is not penetrated as in subcutaneous injection ortransdermal techniques. Optionally, the antigen may pass through thestratum corneum via a hair follicle or a skin organelle (e.g., sweatgland, oil gland).

Transcutaneous immunization with bacterial ADP-ribosylating exotoxins(bARE) as an example, may target the epidermal Langerhans cell, known tobe among the most efficient of the antigen presenting cells (APC).Maturation of APC may be assessed by morphology and phenotype (e.g.,expression of MHC Class II molecules, CD83, or co-stimulatorymolecules). We have found that bARE appear to activate Langerhans cellswhen applied epicutaneously to intact skin. Adjuvants such astrypsin-cleaved bARE may enhance Langerhans cell activation. Langerhanscells direct specific immune responses through phagocytosis of theantigens, and migration to the lymph nodes where they act as APC topresent the antigen to lymphocytes, and thereby induce a potent antibodyresponse. Although the skin is generally considered a barrier topathogens, the imperfection of this barrier is attested to by thenumerous Langerhans cells distributed throughout the epidermis that aredesigned to orchestrate the immune response against organisms invadingthrough the skin. According to Udey (Clin Exp Immunol, 107:s6-s8, 1997):

-   -   Langerhans cells are bone-marrow derived cells that are present        in all mammalian stratified squamous epithelia. They comprise        all of the accessory cell activity that is present in uninflamed        epidermis, and in the current paradigm are essential for the        initiation and propagation of immune responses directed against        epicutaneously applied antigens. Langerhans cells are members of        a family of potent accessory cells (‘dendritic cells’) that are        widely distributed, but infrequently represented, in epithelia        and solid organs as well as in lymphoid tissue.    -   It is now recognized that Langerhans cells (and presumably other        dendritic cells) have a life cycle with at least two distinct        stages. Langerhans cells that are located in epidermis        constitute a regular network of antigen-trapping ‘sentinel’        cells. Epidermal Langerhans cells can ingest particulates,        including microorganisms, and are efficient processors of        complex antigens. However, they express only low levels of MHC        class I and II antigens and costimulatory molecules (ICAM-1,        B7-1 and B7-2) and are poor stimulators of unprimed T cells.        After contact with antigen, some Langerhans cells become        activated, exit the epidermis and migrate to T-cell-dependent        regions of regional lymph nodes where they localize as mature        dendritic cells. In the course of exiting the epidermis and        migrating to lymph nodes, antigen-bearing epidermal Langerhans        cells (now the ‘messengers’) exhibit dramatic changes in        morphology, surface phenotype and function. In contrast to        epidermal Langerhans cells, lymphoid dendritic cells are        essentially non-phagocytic and process protein antigens        inefficiently, but express high levels of MHC class I and class        II antigens and various costimulatory molecules and are the most        potent stimulators of naive T cells that have been identified.”

The potent antigen presenting capability of Langerhans cells can beexploited for transcutaneously-delivered immunogens and vaccines. Animmune response using the skin's immune system may be achieved bydelivering the formulation only to Langerhans cells in the stratumcorneum (i.e., the outermost layer of the skin consisting of cornifiedcells and lipids) and subsequently activating the Langerhans cells totake up antigen, migrate to B-cell follicles and/or T-cell dependentregions, and present the antigen to B and/or T lymphocytes. If antigensother that bARE (e.g., toxin, colonization or virulence factor) are tobe phagocytosed by Langerhans cells, then these antigens could also betransported to the lymph node for presentation to T lymphocytes andsubsequently induce an immune response specific for that antigen. Thus,a feature of TCl is the activation of the Langerhans cell, presumably bybARE or derivatives thereof, chemokines, cytokines, PAMP, or otherLangerhans cell activating substance including contact sensitizers andadjuvants. Increasing the size of the skin population of Langerhanscells or their state of activation would also be expected to enhance theimmune response (e.g., acetone pretreatment). In aged subjects orLangerhans cell-depleted skin (i.e., from UV damage), it may be possibleto replenish the population of Langerhans cells (e.g., tretinoinpretreatment).

Adjuvants such as bARE are known to be highly toxic when injected orgiven systemically. But if placed on the surface of intact skin (i.e.,epicutaneous), they are unlikely to induce systemic toxicity. Thus, thetranscutaneous route may allow the advantage of adjuvant effects withoutsystemic toxicity. A similar absence of toxicity could be expected ifthe skin were penetrated only below the stratum corneum (e.g., near orat the epidermis), but not through the dermis. Thus, the ability toinduce activation of the immune system through the skin confers theunexpected advantage of potent immune responses without systemictoxicity.

The magnitude of the antibody response induced by affinity maturationand isotype switching to predominantly IgG antibodies is generallyachieved with T-cell help, and activation of both Th1 and Th2 pathwaysis suggested by the production of IgG1 and IgG2a. Alternatively, a largeantibody response may be induced by a thymus-independent antigen type 1(TI-1) which directly activates the B lymphocyte or could have similaractivating effects on B lymphocytes such as up-regulation of MHC ClassII, B7, CD40, CD25, and ICAM-1 molecules.

The spectrum of commonly known skin immune responses is represented byatopy and contact dermatitis. Contact dermatitis, a pathogenicmanifestation of Langerhans cell activation, is directed by Langerhanscells which phagocytose antigen, migrate to lymph nodes, presentantigen, and sensitize T lymphocytes that migrate to the skin and causethe intense destructive cellular response that occurs at affected skinsites. Such responses are not generally known to be associated withantigen-specific IgG antibodies. Atopic dermatitis may utilize theLangerhans cell in a similar fashion, but is identified with Th2 cellsand is generally associated with high levels of IgE antibody.

On the other hand, transcutaneous immunization with bARE provides auseful and desirable immune response. There are usually no findingstypical of atopy or contact dermatitis given the high levels of IgG thatare induced. Cholera toxin or E. coli heat-labile enterotoxinepicutaneously applied to skin can achieve immunization in the absenceof lymphocyte infiltration 24, 48 and 120 hours after immunization. Theminor skin reactivity seen in preclinical trials were easily treated.This indicates that Langerhans cells engaged by transcutaneousimmunization as they “comprise all of the accessory cell activity thatis present in uninflamed epidermis, and in the current paradigm areessential for the initiation and propagation of immune responsesdirected against epicutaneously applied antigens” (Udey, 1997). Theuniqueness of the transcutaneous immune response here is also indicatedby the both high levels of antigen-specific IgG antibody, and the typeof antibody produced (e.g., IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA) andgenerally the absence of antigen specific IgE antibody. Transcutaneousimmunization could conceivably occur in tandem with skin inflammation ifsufficient activation of antigen presenting cells and T lymphocytes wereto occur in a transcutaneous response coexisting with atopy or contactdermatitis.

Transcutaneous targeting of Langerhans cells may also be used in tandemwith agents to deactivate all or part of their antigen presentingfunction, thereby modifying immunization or preventing sensitization.Techniques to modulate Langerhans activation or other skin immune cellsinclude, for example, the use of anti-inflammatory steroidal ornon-steroidal agents (NSAID); cyclosporin, FK506, rapamycin,cyclophosphamide, glucocorticoids, or other immunosuppressants;interleukin-10; interleukin-1 monoclonal antibodies (mAB) or solublereceptor antagonists (RA); interleukin-1 converting enzyme (ICE)inhibitors; or depletion via superantigens such as throughStaphylococcal enterotoxin A (SEA) induced epidermal Langerhans celldepletion. Similar compounds may be used to modify the innate responseof Langerhans cells and induce different T-helper responses (Th1 or Th2)or may modulate skin inflammatory responses to decrease potential sideeffects of the immunization. Similarly, lymphocytes may beimmunosuppressed before, during or after immunization by administeringimmunosuppressant separately or by coadministration of immunosuppressantwith the formulation. For example, it may be possible to induce a potentsystemic protective immune responses with agents that would normallyresult in allergic or irritant contact hypersensitivity but addinginhibitors of ICE may alleviate adverse skin reactions.

TCl may be accompished through the ganglioside GM1 binding activity ofCT, LT, or subunits thereof (e.g., CTB or LTB). Ganglioside GM1 is aubiquitous cell membrane glycolipid found in all mammalian cells. Whenthe pentameric CT B subunit binds to the cell surface, a hydrophilicpore is formed which allows the A subunit to insert across the lipidbilayer. Other binding targets on the APC may be utilized. The LT Bsubunit binds to ganglioside GM1 in addition to other gangliosides andits binding activities may account for its the fact that LT is highlyimmunogenic on the skin.

TCl with bARE or B subunit-containing fragments or conjugates thereofmay require their GM1 ganglioside binding activity. When mice weretranscutaneously immunized with CT, CTA and CTB, CT and CTB wererequired for induction of an immune response. CTA contains theADP-ribosylating exotoxin activity but only CT and CTB containing thebinding activity are able to induce an immune response indicating thatthe B subunit was necessary and sufficient to immunize through the skin.We conclude that the Langerhans cells or other APC may be activated byCTB binding to its cell surface resulting in a transcutaneous immuneresponse.

Antigen

A transcutaneous immunization system delivers agents to specializedcells (e.g., antigen presentation cell, lymphocyte) that produce animmune response. These agents as a class are called antigens. Antigenmay be composed of chemical structures such as, for example,carbohydrate, glycolipid, glycoprotein, lipid, lipoprotein,phospholipid, polypeptide, conjugates thereof, or any other materialknown to induce an immune response. Antigen may be provided as a wholeorganism such as, for example, a bacterium or virion; antigen may beobtained from an extract or lysate, either from whole cells or membranealone; or antigen may be chemically synthesized or produced byrecombinant technology.

Antigen of the invention may be expressed by recombinant technology,preferably as a fusion with an affinity or epitope tag; chemicalsynthesis of an oligopeptide, either free or conjugated to carrierproteins, may be used to obtain antigen of the invention. Oligopeptidesare considered a type of polypeptide. Oligopeptide lengths of 6 residuesto 20 residues are preferred. Polypeptides may also by synthesized asbranched structures (e.g., U.S. Pat. Nos. 5,229,490 and 5,390,111).Antigenic polypeptides include, for example, synthetic or recombinantB-cell and T-cell epitopes, universal T-cell epitopes, and mixed T-cellepitopes from one organism or disease and B-cell epitopes from another.Antigen obtained through recombinant technology or peptide synthesis, aswell as antigen obtained from natural sources or extracts, may bepurified by the antigen's physical and chemical characteristics,preferably by fractionation or chromatography. Recombinants may combineB subunits or chimeras of bARE. A multivalent antigen formulation may beused to induce an immune response to more than one antigen at the sametime. Conjugates may be used to induce an immune response to multipleantigens, to boost the immune response, or both. Additionally, toxinsmay be boosted by the use of toxoids, or toxoids boosted by the use oftoxins. Transcutaneous immunization may be used to boost responsesinduced initially by other routes of immunization such as by oral, nasalor parenteral routes. Antigen includes, for example, toxins, toxoids,subunits thereof, or combinations thereof (e.g., cholera toxin, tetanustoxoid); additionally, toxins, toxoids, subunits thereof, orcombinations thereof may act as both antigen and adjuvant. Suchoral/transcutaneous or transcutaneous/oral immunization may beespecially important to enhance mucosal immunity in diseases wheremucosal immunity correlates with protection.

Antigen may be solubilized in a buffer or water or organic solvents suchas alcohol or DMSO, or incorporated in gels, emulsion, microemulsions,and creams. Suitable buffers include, but are not limited to, phosphatebuffered saline Ca⁺⁺/Mg⁺⁺ free, phosphate buffered saline, normal saline(150 mM NaCl in water), and Hepes or Tris buffer. Antigen not soluble inneutral buffer can be solubilized in 10 mM acetic acid and then dilutedto the desired volume with a neutral buffer such as PBS. In the case ofantigen soluble only at acid pH, acetate-PBS at acid pH may be used as adiluent after solubilization in dilute acetic acid. Glycerol may be asuitable non-aqueous buffer for use in the invention.

A hydrophobic antigen can be solubilized in a detergent or surfactant,for example a polypeptide containing a membrane-spanning domain.Furthermore, for formulations containing liposomes, an antigen in adetergent solution (e.g., cell membrane extract) may be mixed withlipids, and liposomes then may be formed by removal of the detergent bydilution, dialysis, or column chromatography. Certain antigens (e.g.,membrane proteins) need not be soluble per se, but can be inserteddirectly into a lipid membrane (e.g., a virosome), in a suspension ofvirion alone, or suspensions of microspheres or heat-inactivatedbacteria which may be taken up by activate antigen presenting cells(e.g., opsonization). Antigens may also be mixed with a penetrationenhancer as described in WO 99/43350.

Many antigens are known in the art which can be used to vaccinate humanor animal subjects and induce an immune response specific for particularpathogens, as well as methods of preparing antigen, determining asuitable dose of antigen, assaying for induction of an immune response,and treating infection by a pathogen (e.g., bacterium, virus, fungus, orprotozoan).

The effect of Escherichia coli infection of mammals is dependent on theparticular strain of organism. Many beneficial E. coli are present inthe intestines. Since the initial association with diarrheal illness,five categories of diarrheagenic E. coli have been identified:enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic(EHEC), enteroaggregative (EAggEC), and enteroinvasive (EIEC). They aregrouped according to characteristic virulence properties, such aselaboration of toxins and colonization factors and/or by specific typesof interactions with intestinal epithelial cells. ETEC are the mostcommon of the diarrheagenic E. coli and pose the greatest risk totravelers. Strains which have been cultured from humans include B7A(CS6, LT, STa), H10407 (CFA/I, LT, STa) and E24377A (CS3, CS1, LT, STa).They may be used singly or in combination as whole-cell sources ofantigen providing a variety of different toxins and colonizationfactors.

There is a need for vaccines which are specific against enterotoxigenicE. coli that give rise to antibodies that cross-react with andcross-protect against the more common colonization and virulencefactors. The CS4-CFA/I family of fimbrial proteins are found on some ofthe more prevalent enterotoxigenic E. coli strains: there are sixmembers of this family of ETEC antigens, CFA/I, CS1, CS2, CS4, CS17, andPCF 0166.

Colonization factor antigens (CFA) of ETEC are important in the initialstep of colonization and adherence of the bacterium to intestinalepithelia. In epidemiological studies of adults and children withdiarrhea, CFA/I is found in a large percentage of morbidity attributedto ETEC. The CFA/I is present on the surfaces of bacteria in the form ofpili (fimbriae), which are rigid, 7 nm diameter protein fibers composedof repeating pilin subunits. The CFA/I antigens promotemannose-resistant attachment to human brush borders with an apparentsialic acid sensitivity. Hence, it has been postulated that a vaccinethat establishes immunity against these proteins may prevent attachmentto host tissues and subsequent disease.

Other antigens including CS3, CS5, and CS6. CFA/I, CS3 and CS6 may occuralone, but with rare exception CS1 is only found with CS3, CS2 with CS3,CS4 with CS6 and CS5 with CS6. Serological studies show these antigensoccur in strains accounting for up to about 75% or as little as about25% of ETEC cases, depending on the location of the study.

Consensus peptides have been described in U.S. Pat. No. 5,914,114 whichraise antibodies against the antigens of all members of the E. colifamily CS4-CFA/I. While the N-terminus of members of this family shows ahigh degree of identity, the remainder of the sequence of the proteinsshows less relatedness across the strains. Consensus peptides encompassknown linear B- and T-cell epitopes, and bears a high degree ofevolutionary relatedness across the six different colonization factors.For example, consensus peptides have the amino acid sequence (an aminoacid residue may be added to either termini or modified internally toprovide a reactive linkage): VEKNITVTASVDPTIDLLQADGSALPSAVALTYSPA (SEQID NO: 1) and VEKNITVTASVDPTIDLLQADGSALPASVALTYSPA (SEQ ID NO: 2).

These consensus peptides were constructed based on the homologousregions of the CFA/I, CS1, CS2, CS4, CS17, and PCF 0166 antigens.

TABLE 1 Alignment of antigens of the CS4-CFA/I family (SEQ ID NOS: 3-8)Antigen Amino Acid Sequence CFA/I VEKNITVTASVDPVIDLLQADGSALPSAVALTYSPASCS1 VEKTISVTASVDPTVDLLQSDGSALPNSVALTYSPAV CS2 AEINITVTASVDPVIDLLQA CS4VEKNITVTASVDPTIDILQADGSYLPTAVELTYSPAA CS17VEKNITVRASVDKLIDLLQADGTSLPDSIALTYSVA PCF0166 VEKNITVTASVDPTIDILQANGSAL

CS6, a component of colonization factor IV (CFA/IV), can also be foundin more than about 25% of ETEC strains in serological surveys (e.g.,soldiers in the Middle East). The nucleotide sequences of CS3 and CS6antigens, along with a process for producing them, are described in U.S.Pat. No. 5,698,416.

Other antigens which may be used are toxins that cause enteric diseasesuch as, for example, shiga toxin and E. coli enterotoxins. Heat-labileenterotoxin (LT) is described below, but heat-stable enterotoxins (e.g.,STa, STb) which cause disease symptoms may also be neutralized byantibody. LT is a periplasmic toxin and ST is an extracellular toxin.STa is methanol soluble and STb is methanol insoluble. Two differentprecursors are used: STa is a 18-19 amino acid peptide and STb is a 48amino acid peptide with no sequence similarity between them. Conjugatesbetween LT and ST or ST multimers may also be used (see U.S. Pat. No.4,886,663).

It would be advantageous for a vaccine to be developed for a broad rangeof common traveler's diseases, especially enteric infectious diseases.For example, campylobacteriosis (Campylobacter jejuni), giardiasis(Giardia intestinalis), hepatitis (hepatitis virus A or B), malaria(Plasmodium falciparum, P. vivax, P. ovale, and P. malarae), shigellosis(Shigella boydii, S. dysenterae, S. flexneri, and S. sonnet), viralgastroenteritis (rotavirus), and combinations thereof may be treated byincluding antigens derived from the responsible pathogen. Systemic ormucosal antibodies that neutralize toxicity or block attachment andentry into the cell are desirable. An immune response which is specificfor molecules associated with pathogens (e.g., toxins, membraneproteins) may be induced by various routes of administration (e.g.,enteral, mucosal, parenteral, transcutaneous).

Adjuvant

The formulation contains an adjuvant, although a single molecule maycontain both adjuvant and antigen properties (e.g., E. coli heat-labileenterotoxin). Adjuvants are substances that are used to specifically ornon-specifically potentiate an antigen-specific immune response, perhapsthrough activation of antigen presenting cells (e.g., dendritic cells invarious layers of the skin, especially Langerhans cells). See also Elsonet al. (in Handbook of Mucosal Immunology, Academic Press, 1994).Although activation may initially occur in the epidermis or dermis, theeffects may persist as the dendritic cells migrate through the lymphsystem and the circulation. Adjuvant may be formulated and applied withor without antigen, but generally, activation of antigen presentingcells by adjuvant occurs prior to presentation of antigen.Alternatively, they may be separately presented within a short intervalof time but targeting the same anatomical region (e.g., the samedraining lymph node field).

Adjuvants include, for example, chemokines (e.g., defensins, HCC-1,HCC4, MCP-1, MCP-3, MCP4, MIP-1α, MIP-1β, MIP-1δ, MIP-3α, MIP-2,RANTES); other ligands of chemokine receptors (e.g., CCR1, CCR-2, CCR-5,CCR-6, CXCR-1); cytokines (e.g., IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12;IFN-γ; TNF-α; GM-CSF); other ligands of receptors for those cytokines,immunostimulatory CpG motifs in bacterial DNA or oligonucleotides;muramyl dipeptide (MDP) and derivatives thereof (e.g., murabutide,threonyl-MDP, muramyl tripeptide); heat shock proteins and derivativesthereof; Leishmania homologs of elF4a and derivatives thereof; bacterialADP-ribosylating exotoxins and derivatives thereof (e.g., geneticmutants, A and/or B subunit-containing fragments, chemically toxoidedversions); chemical conjugates or genetic recombinants containingbacterial ADP-ribosylating exotoxins or derivatives thereof; C3d tandemarray; lipid A and derivatives thereof (e.g., monophosphoryl ordiphosphoryl lipid A, lipid A analogs, AGP, AS02, AS04, DC-Chol, Detox,OM-174); ISCOMS and saponins (e.g., QUIL A, QS-21); squalene;superantigens; or salts (e.g., aluminum hydroxide or phosphate, calciumphosphate). See also Nohria et al. (Biotherapy, 7:261-269, 1994) andRichards et al. (in Vaccine Design, Eds. Powell et al., Plenum Press,1995) for other useful adjuvants.

Adjuvant may be chosen to preferentially induce antibody or cellulareffectors, specific antibody isotypes (e.g., IgM, IgD, IgA1, IgA2,secretory IgA, IgE, IgG1, IgG2, IgG3, and/or IgG4), or specific T-cellsubsets (e.g., CTL, Th1, Th2 and/or T_(DTH)). For example, antigenpresenting cells may present Class II-restricted antigen to precursorCD4+ T cells, and the Th1 or Th2 pathway may be entered. T helper cellsactively secreting cytokine are primary effector cells; they are memorycells if they are resting. Reactivation of memory cells produces memoryeffector cells. Th1 characteristically secrete IFN-γ (TNF-β and IL-2 mayalso be secreted) and are associated with “help” for cellular immunity,while Th2 characteristically secrete IL-4 (IL-5 and IL-13 may also besecreted) and are associated with “help” for humoral immunity. Dependingon disease pathology, adjuvants may be chosen to prefer a Th1 response(e.g., antigen-specific cytolytic cells) vs. a Th2 response (e.g.,antigen-specific antibodies).

Unmethylated CpG dinucleotides or similar motifs are known to activate Blymphocytes and macrophages (see U.S. Pat. No. 6,218,371). Other formsof bacterial DNA can be used as adjuvants. Bacterial DNA is among aclass of structures which have patterns allowing the immune system torecognize their pathogenic origins to stimulate the innate immuneresponse leading to adaptive immune responses. These structures arecalled pathogen-associated molecular patterns (PAMP) and includelipopolysaccharides, teichoic acids, unmethylated CpG motifs,double-stranded RNA, and mannins. PAMP induce endogenous signals thatcan mediate the inflammatory response, act as costimulators of T-cellfunction and control the effector function. The ability of PAMP toinduce these responses play a role in their potential as adjuvants andtheir targets are antigen presenting cells such as dendritic cells andmacrophages. The antigen presenting cells of the skin could likewise bestimulated by PAMP transmitted through the skin. For example, Langerhanscells, a type of dendritic cell, could be activated by PAMP in solutionon the skin with a transcutaneously poorly immunogenic molecule and beinduced to migrate and present this poorly immunogenic molecule toT-cells in the lymph node, inducing an antibody response to the poorlyimmunogenic molecule. PAMP could also be used in conjunction with otherskin adjuvants such as cholera toxin to induce different costimulatorymolecules and control different effector functions to guide the immuneresponse, for example from a Th2 to a Th1 response.

Most ADP-ribosylating exotoxins (bARE) are organized as A:B heterodimerswith a B subunit containing the receptor binding activity and an Asubunit containing the ADP-ribosyltransferase activity. Exemplary bAREinclude cholera toxin (CT) E. coli heat-labile enterotoxin (LT),diphtheria toxin, Pseudomonas exotoxin A (ETA), pertussis toxin (PT), C.botulinum toxin C2, C. botulinum toxin C3, C. limosum exoenzyme, B.cereus exoenzyme, Pseudomonas exotoxin S, S. aureus EDIN, and B.sphaeticus toxin. Mutant bARE, for example containing mutations of thetrypsin cleavage site (e.g., Dickenson et al., Infect Immun,63:1617-1623, 1995) or mutations affecting ADP-ribosylation (e.g., Douceet al., Infect Immun, 65:28221-282218, 1997) may be used.

CT, LT, ETA and PT, despite having different cellular binding sites, arepotent adjuvants for transcutaneous immunization, inducing IgGantibodies but not IgE antibodies. CTB without CT can also induce IgGantibodies. Thus, both bARE and a derivative thereof can effectivelyimmunize when epicutaneously applied to the skin. Native LT as anadjuvant and antigen, however, is clearly not as potent as native CT.But activated bARE can act as adjuvants for weakly immunogenic antigensin a transcutaneous immunization system. Thus, therapeutic immunizationwith one or more antigens could be used separately or in conjunctionwith immunostimulation of the antigen presenting cell to induce aprophylactic or therapeutic immune response.

In general, toxins can be chemically inactivated to form toxoids whichare less toxic but remain immunogenic. We envision that thetranscutaneous immunization system using toxin-based immunogens andadjuvants can achieve anti-toxin levels adequate for protection againstthese diseases. The anti-toxin antibodies may be induced throughimmunization with the toxins, or genetically-detoxified toxoidsthemselves, or with toxoids and adjuvants. Genetically toxoided toxinswhich have altered ADP-ribosylating exotoxin activity or trypsincleavage site, but not binding activity, are envisioned to be especiallyuseful as non-toxic activators of antigen presenting cells used intranscutaneous immunization and may reduce concerns over toxin use.

bARE can also act as an adjuvant to induce antigen-specific CTL throughtranscutaneous immunization. The bARE adjuvant may be chemicallyconjugated to other antigens including, for example, carbohydrates,polypeptides, glycolipids, and glycoprotein antigens. Chemicalconjugation with toxins, their subunits, or toxoids with these antigenswould be expected to enhance the immune response to these antigens whenapplied epicutaneously. To overcome the problem of the toxicity of thetoxins (e.g., diphtheria toxin is known to be so toxic that one moleculecan kill a cell) and to overcome the problems of working with suchpotent toxins as tetanus, several workers have taken a recombinantapproach to producing genetically-produced toxoids. This is based oninactivating the catalytic activity of the ADP-ribosyl transferase bygenetic deletion. These toxins retain the binding capabilities, but lackthe toxicity, of the natural toxins. Such genetically toxoided exotoxinswould be expected to induce a transcutaneous immune response and to actas adjuvants. They may provide an advantage in a transcutaneousimmunization system in that they would not create a safety concern asthe toxoids would not be considered toxic. Activation through atechnique such as trypsin cleavage, however, would be expected toenhance the adjuvant qualities of LT through the skin which lackstrypsin-like enzymes. Additionally, several techniques exist tochemically modify toxins and can address the same problem. Thesetechniques could be important for certain applications, especiallypediatric applications, in which ingested toxins might possibly createadverse reactions.

Adjuvant may be biochemically purified from a natural source (e.g., pCTor pLT) or recombinantly produced (e.g., rCT or rLT). ADP-ribosylatingexotoxin may be purified either before or after proteolysis (i.e.,activation). B subunit of the ADP-ribosylating exotoxin may also beused: purified from the native enzyme after proteolysis or produced froma fragment of the entire coding region of the enzyme. The subunit of theADP-ribosylating exotoxin may be used separately (e.g., CTB or LTB) ortogether (e.g., CTA-LTB, LTA-CTB) by chemical conjugation or geneticfusion.

Point mutations (e.g., single, double, or triple amino acidsubstitutions), deletions (e.g., protease recognition site), andisolated functional domains of ADP-ribosylating exotoxin may also beused as adjuvant. Derivatives which are less toxic or have lost theirADP-ribosylation activity, but retain their adjuvant activity have beendescribed. Specific mutants of E. coli heat-labile enterotoxin includeLT-K63, LT-R72, LT (H44A), LT (R192G), LT (R192G/L211A), and LT(Δ192-194). Toxicity may be assayed with the Y-1 adrenal cell assay(Clements and Finkelstein, Infect Immun, 24:760-769, 1979).ADP-ribosylation may be assayed with the NAD-agmatineADP-ribosyltransferase assay (Moss et al., J Biol Chem, 268:6383-6387,1993). Particular ADP-ribosylating exotoxins, derivatives thereof, andprocesses for their production and characterization are described inU.S. Pat. Nos. 4,666,837; 4,935,364; 5,308,835; 5,785,971; 6,019,982;6,033,673; and 6,149,919.

An activator of Langerhans cells may also be used as an adjuvant.Examples of such activators include: inducers of heat shock protein;contact sensitizers (e.g., trinitrochlorobenzene, dinitrofluorobenzene,nitrogen mustard, pentadecylcatechol); toxins (e.g., Shiga toxin, Staphenterotoxin B); lipopolysaccharide (LPS), lipid A, or derivativesthereof; bacterial DNA; cytokines (e.g., TNF-α, IL-1β, IL-10, IL-12);members of the TGFβ superfamily, calcium ions in solution, calciumionophores, and chemokines (e.g., defensins 1 or 2, RANTES, MIP-1α,MIP-2, IL-8).

If an immunizing antigen has sufficient Langerhans cell activatingcapabilities then a separate adjuvant may not be required, as in thecase of LT which is both antigen and adjuvant. Alternatively, suchantigens can be considered not to require an adjuvant because they aresufficiently immunogenic. It is envisioned that live cell or viruspreparations, attenuated live cells or viruses, killed cells,inactivated viruses, and DNA plasmids could be effectively used fortranscutaneous immunization. It may also be possible to use lowconcentrations of contact sensitizers or other activators of Langerhanscells to induce an immune response without inducing skin lesions.

Other techniques for enhancing activity of adjuvants may be effective,such as adding surfactants and/or phospholipids to the formulation toenhance adjuvant activity of ADP-ribosylating exotoxin byADP-ribosylation factor. One or more ADP-ribosylation factors (ARF) maybe used to enhance the adjuvanticity of bARE (e.g., ARF1, ARF2, ARF3,ARF4, ARF5, ARF6, ARD1). Similarly, one or more ARF could be used withan ADP-ribosylating exotoxin to enhance its adjuvant activity.

Undesirable properties or harmful side effects (e.g., allergic orhypersensitive reaction; atopy, contact dermatitis, or eczema; systemictoxicity) may be reduced by modification without destroying itseffectiveness in transcutaneous immunization. Modification may involve,for example, removal of a reversible chemical modification (e.g.,proteolysis) or encapsulation in a coating which reversibly isolates oneor more components of the formulation from the immune system. Forexample, one or more components of the formulation may be encapsulatedin a particle for delivery (e.g., microspheres, nanoparticles) althoughwe have shown that encapsulation in lipid vesicles is not required fortranscutaneous immunization and appears to have a negative effect.Phagocytosis of a particle may, by itself, enhance activation of anantigen presenting cell by upregulating expression of MHC Class I and/orClass II molecules and/or costimulatory molecules (e.g., CD40, B7 familymembers like CD80 and CD86).

Formulation

Processes for manufacturing a pharmaceutical formulation are well known.The components of the formulation may be combined with apharmaceutically-acceptable carrier or vehicle, as well as anycombination of optional additives (e.g., at least one diluent, binder,excipient, stabilizer, dessicant, preservative, coloring, orcombinations thereof. See, generally, Ullmann's Encyclopedia ofIndustrial Chemistry, 6^(th) Ed. (electronic edition, 1998); Remington'sPharmaceutical Sciences, 22^(nd) (Gennaro, 1990, Mack Publishing);Pharmaceutical Dosage Forms, 2^(nd) Ed. (various editors, 1989-1998,Marcel Dekker); and Pharmaceutical Dosage Forms and Drug DeliverySystems (Ansel et al., 1994, Williams & Wilkins).

Good manufacturing practices are known in the pharmaceutical industryand regulated by government agencies (e.g., Food and DrugAdministration). Sterile liquid formulations may be prepared bydissolving an intended component of the formulation in a sufficientamount of an appropriate solvent, followed by sterilization byfiltration to remove contaminating microbes. Generally, dispersions areprepared by incorporating the various sterilized components of theformulation into a sterile vehicle which contains the basic dispersionmedium. For production of solid forms that are required to be sterile,vacuum drying or freeze drying can be used. Solid dosage forms (e.g.,powders, granules, pellets, tablets) or liquid dosage forms (e.g.,liquid in ampules, capsules, vials) can be made from at least one activeingredient or component of the formulation.

Suitable procedures for making the various dosage forms and productionof patches are known. The formulation may also be produced byencapsulating solid or liquid forms of at least one active ingredient orcomponent, or keeping them separate in compartments or chambers. Thepatch may include a compartment containing a vehicle (e.g., a salinesolution) which is disrupted by pressure and subsequently solubilizesthe dry formulation of the patch. The size of each dose and the intervalof dosing to the subject may be used to determine a suitable size andshape of the container, compartment, or chamber.

Formulations will contain an effective amount of the active ingredients(e.g., antigen and adjuvant) together with carrier or suitable amountsof vehicle in order to provide pharmaceutically-acceptable compositionssuitable for administration to a human or animal. Formulation thatinclude a vehicle may be in the form of an cream, emulsion, gel, lotion,ointment, paste, solution, suspension, or other liquid forms known inthe art; especially those that enhance skin hydration.

The relative amounts of active ingredients within a dose and the dosingschedule may be adjusted appropriately for efficacious administration toa subject (e.g., animal or human). This adjustment may depend on thesubject's particular disease or condition, and whether therapy orprophylaxis is intended. To simplify administration of the formulationto the subject, each unit dose would contain the active ingredients inpredetermined amounts for a single round of immunization.

There are numerous causes of protein instability or degradation,including hydrolysis and denaturation. In the case of denaturation, theprotein's conformation is disturbed and the protein may unfold from itsusual globular structure. Rather than refolding to its naturalconformation, hydrophobic interaction may cause clumping of moleculestogether (i.e., aggregation) or refolding to an unnatural conformation.Either of these results may entail diminution or loss of antigenic oradjuvant activity. Stabilizers may be added to lessen or prevent suchproblems.

The formulation, or any intermediate in its production, may bepretreated with protective agents (i.e., cryoprotectants and drystabilizers) and then subjected to cooling rates and final temperaturesthat minimize ice crystal formation. By proper selection ofcryoprotective agents and the use of preselected drying parameters,almost any formulation might be cryoprepared for a suitable desired enduse.

It should be understood in the following discussion of optionaladditives like excipients, stabilizers, dessicants, and preservativesare described by their function. Thus, a particular chemical may act assome combination of excipient, stabilizer, dessicant, and/orpreservative. Such chemicals would be consideredimmunologically-inactive because it does not directly induce an immuneresponse, but it increases the response by enhancing immunologicalactivity of the antigen or adjuvant: for example, by reducingmodification of the antigen or adjuvant, or denaturation during dryingand dissolving cycles.

Stabilizers include cyclodextrin and derivatives thereof (see U.S. Pat.No. 5,730,969). Suitable preservatives such as sucrose, mannitol,sorbitol, trehalose, dextran, and glycerin can also be added tostabilize the final formulation. A stabilizer selected from nonionicsurfactants, D-glucose, D-galactose, D-xylose, D-glucuronic acid, saltsof D-glucuronic acid, trehalose, dextrans, hydroxyethyl starches, andmixtures thereof may be added to the formulation. Addition of an alkalimetal salt or magnesium chloride may stabilize a polypeptide, optionallyincluding serum albumin and freeze-drying to further enhance stability.A polypeptide may also be stabilized by contacting it with a saccharideselected from the group consisting of dextran, chondroitin sulfuricacid, starch, glycogen, insulin, dextrin, and alginic acid salt. Othersugars that can be added include monosaccharides, disaccharides, sugaralcohols, and mixtures thereof (e.g., glucose, mannose, galactose,fructose, sucrose, maltose, lactose, mannitol, xylitol). Polyols maystabilize a polypeptide, and are water-miscible or water-soluble.Suitable polyols may be polyhydroxy alcohols, monosaccharides anddisaccharides including mannitol, glycerol, ethylene glycol, propyleneglycol, trimethyl glycol, vinyl pyrrolidone, glucose, fructose,arabinose, mannose, maltose, sucrose, and polymers thereof. Variousexcipients may also stabilize polpeptides, including serum albumin,amino acids, heparin, fatty acids and phospholipids, surfactants,metals, polyols, reducing agents, metal chelating agents, polyvinylpyrrolidone, hydrolyzed gelatin, and ammonium sulfate.

Single-dose formulations can be stabilized in poly(lactic acid) (PLA)and poly (lactide-co-glycolide) (PLGA) microspheres by suitable choiceof excipient or stabilizer. Trehalose may be advantageously used as anadditive because it is a non-reducing saccharide, and therefore does notcause aminocarbonyl reactions with substances bearing amino groups suchas proteins.

It is conceivable that a formulation that can be administered to thesubject in a dry, non-liquid form, may allow storage in conditions thatdo not require a cold chain. An antigen (e.g., CS6) in solution may bemixed in solution with an adjuvant such as LT and is placed on a gauzepad with an occlusive backing such as plastic wrap and allowed to dry.This patch can then be placed on skin with the gauze side in directcontact with the skin for a period of time and can be held in placecovered with a simple occlusive such as plastic wrap and adhesive tape.The patch may have many compositions. The substrate may be cotton gauze,combinations of rayon-nylon or other synthetic materials and may haveocclusive solid backings including polyvinyl chloride, rayons, otherplastics, gels, creams, emulsions, waxes, oils, parafilm, rubbers(synthetic or natural), cloths, or membranes. The patch can be held ontothe skin and components of the patch can be held together using variousadhesives. One or more of the adjuvant and/or antigen may beincorporated into the substrate or adhesive parts of the patch.

A liquid or quasi-liquid formulation may be applied directly to the skinand allowed to air dry; rubbed into the skin or scalp; placed on theear, inguinal, or intertriginous regions, especially in animals; placedon the anal/rectal tissues; held in place with a dressing, patch, orabsorbent material; immersion; otherwise held by a device such as astocking, slipper, glove, or shirt; or sprayed onto the skin to maximizecontact with the skin. The formulation may be applied in an absorbentdressing or gauze. The formulation may be covered with an occlusivedressing such as, for example, AQUAPHOR (an emulsion of petrolatum,mineral oil, mineral wax, wool wax, panthenol, bisabol, and glycerinfrom Beiersdorf), plastic film, COMFEEL (Coloplast) or VASELINEpetroleum jelly; or a non-occlusive dressing such as, for example,TEGADERM (3M), DUODERM (3M) or OPSITE (Smith & Napheu). An occlusivedressing excludes the passage of water. Such a formulation may beapplied to single or multiple sites, to single or multiple limbs, or tolarge surface areas of the skin by complete immersion. The formulationmay be applied directly to the skin. Other substrates that may be usedare pressure-sensitive adhesives such as acrylics, polyisobutylenes, andsilicones. The formulation may be incorporated directly into suchsubstrates, perhaps with the adhesive per se instead of adsorption to aporous pad (e.g., gauze) or bilious strip (e.g., filter paper).

Whether or not a patch is used, polymers added to the formulation mayact as an excipient, stabilizer, and/or preservative of an activeingredient as well as reducing the concentration of the activeingredient that saturates a solution used to dissolve a dry form of theactive ingredient. Such reduction occurs because the polymer reduces theeffective volume of the solution by filling the “empty” space. In thisway, quantities of antigen/adjuvant can be conserved without reducingthe amount of saturated solution. An important thermodynamicconsideration is that an active ingredient in the saturated solutionwill be “driven” into regions of lower concentration (e.g., through theskin). In solution, polymers can also stabilize and/or preserve theantigen/adjuvant-activity of solubilized ingredients of the formulation.Such polymers include ethylene or propylene glycol, vinyl pyrrolidone,and β-cyclodextrin polymers and copolymers.

Transcutaneous Delivery

Transcutaneous delivery of the formulation may target Langerhans cellsand, thus, achieve effective and efficient immunization. These cells arefound in abundance in the skin and are efficient antigen presentingcells (APC), which can lead to T-cell memory and potent immuneresponses. Because of the presence of large numbers of Langerhans cellsin the skin, the efficiency of transcutaneous delivery may be related tothe surface area exposed to antigen and adjuvant. In fact, the reasonthat transcutaneous immunization is so efficient may be that it targetsa larger number of these efficient antigen presenting cells thanintramuscular immunization.

The invention will enhance access to immunization, while inducing apotent immune response. Because transcutaneous immunization does notrequire injection with a hypodermic needle (i.e., penetration to orthrough the dermis) and the complications and difficulties thereof, therequirements of medically-sophisticated personnel, sterile technique,and sterile equipment are reduced. Furthermore, the barriers toimmunization at multiple sites or to multiple immunizations arediminished. Immunization by a single application of the formulation isalso envisioned.

Immunization may be achieved using topical or epicutaneous applicationof a simple formulation of antigen and adjuvant, optionally covered byan occlusive dressing or using other patch technologies, to intact skinwith or without chemical or physical penetration. The immunization couldbe given by untrained personnel, and is amenable to self-application.Large-scale field immunization could occur given the easy accessibilityto immunization. Additionally, a simple immunization procedure wouldimprove access to immunization by pediatric, elderly, and Third Worldpopulations. Transcutaneous immunization according to the invention mayprovide a method whereby antigens and adjuvant can be delivered to theimmune system, especially specialized antigen presentation cellsunderlying the skin (e.g., Langerhans cells).

For traditional vaccines, their formulations were injected through theskin with needles. Injection of vaccines using needles carries certaindrawbacks including the need for sterile needles and syringes, trainedmedical personnel to administer the vaccine, discomfort from theinjection, needle-born diseases, and potential complications broughtabout by puncturing the skin with the potentially reusable needles.Immunization through the skin without the use of hypodermic needlesrepresents an advance for vaccine delivery by avoiding the hypodermicneedles.

Moreover, transcutaneous immunization may be superior to immunizationusing hypodermic needles as more immune cells would be targeted by theuse of several locations targeting large surface areas of skin. Atherapeutically-effective amount of antigen sufficient to induce animmune response may be delivered transcutaneously either at a singlecutaneous location, or over an area of skin covering multiple draininglymph node fields (e.g., cervical, axillary, inguinal, epitrochelear,popliteal, those of the abdomen and thorax). Such locations close tonumerous different lymphatic nodes at locations all over the body willprovide a more widespread stimulus to the immune system than when asmall amount of antigen is injected at a single location by intradermal,subcutaneous, or intramuscular injection.

Antigen passing through or into the skin may encounter antigenpresenting cells which process the antigen in a way that induces animmune response. Multiple immunization sites may recruit a greaternumber of antigen presenting cells and the larger population of antigenpresenting cells that were recruited would result in greater inductionof the immune response. It is conceivable that use of the skin maydeliver antigen to phagocytic cells of the skin such as, for example,dendritic cells, Langerhans cells, macrophages, and other skin antigenpresenting cells; antigen may also be delivered to phagocytic cells ofthe liver, spleen, and bone marrow that are known to serve as theantigen presenting cells through the blood stream or lymphatic system.

Langerhans cells, other dendritic cells, macrophages, or combinationsthereof may be specifically targeted using their asialoglycoproteinreceptor, mannose receptor, Fcγ receptor CD64, high-affinity receptorfor IgE, or other highly expressed membrane proteins. A ligand orantibody specific for any of those receptors may be conjugated to orrecombinantly produced as a protein fusion with adjuvant, antigen, orboth. Furthermore, adjuvant, antigen, or both may be conjugated to orrecombinantly produced as a protein fusion with protein A or protein Gto target surface immunoglobulin of B lymphocytes. The envisioned resultwould be widespread distribution of antigen to antigen presenting cellsto a degree that is rarely, if ever achieved, by current immunizationpractices.

Genetic immunization has been described in U.S. Pat. Nos. 5,589,466,5,593,972, and 5,703,055. The nucleic acid(s) contained in theformulation may encode the antigen, the adjuvant, or both. The nucleicacid may or may not be capable of replication; it may be non-integratingand non-infectious. For example, the nucleic acid may encode a fusionpolypeptide comprising antigen and a ubiquitin domain to direct theimmune response to a class I restricted response. The nucleic acid mayfurther comprise a regulatory region operably linked to the sequenceencoding the antigen or adjuvant. The nucleic acid may be added with anadjuvant. The nucleic acid may be complexed with an agent that promotestransfection such as cationic lipid, calcium phosphate, DEAE-dextran,polybrene-DMSO, or a combination thereof. Immune cells can be targetedby conjugation of DNA to Fc receptor or protein A/G, or attaching DNA toan agent linking it to α₂-macroglobulin or protein A/G or similar APCtargeting material.

A specific immune response may comprise humoral (i.e., antigen-specificantibody) and/or cellular (i.e., antigen-specific lymphocytes such as Blymphocytes, CD4⁺T cells, CD8⁺T cells, CTL, Th1 cells, Th2 cells, and/orT_(DTH) cells) effector arms. Moreover, the immune response may compriseNK cells and other leukocytes that mediate antibody-dependentcell-mediated cytotoxicity (ADCC).

The immune response induced by the formulation of the invention mayinclude the elicitation of antigen-specific antibodies and/orlymphocytes. Antibody can be detected by immunoassay techniques.Detection of the various antibody isotypes (e.g., IgM, IgD, IgA1, IgA2,secretory IgA, IgE, IgG1, IgG2, IgG3, or IgG4) can be indicative of asystemic or regional immune response. Immune responses can also bedetected by a neutralizing assay. Antibodies are protective proteinsproduced by B lymphocytes. They are highly specific, generally targetingone epitope of an antigen. Often, antibodies play a role in protectionagainst disease by specifically reacting with antigens derived from thepathogens causing the disease. Immunization may induce antibodiesspecific for the immunizing antigen (e.g., bacterial toxin).

CTL are immune cells produced to protect against infection by apathogen. They are also highly specific. Immunization may induce CTLspecific for the antigen, such as a synthetic oligopeptide based on amalaria protein, in association with self-major histocompatibilityantigen. CTL induced by immunization with the transcutaneous deliverysystem may kill pathogen-infected cells. Immunization may also produce amemory response as indicated by boosting responses in antibodies andCTL, lymphocyte proliferation by culture of lymphocytes stimulated withthe antigen, and delayed type hypersensitivity responses to intradermalskin challenge of the antigen alone.

Successful protection could also be demonstrated by challenge studiesusing infection by the pathogen or administration of toxin, ormeasurement of a clinical criterion (e.g., high antibody titers orproduction of IgA antibody-secreting cells in mucosal membranes may beused as a surrogate marker).

The following is meant to be illustrative of the invention, but practiceof the invention is not limited or restricted in any way by thefollowing examples.

ANIMAL EXAMPLES

BALB/c and C57BL/6 mice were obtained from Jackson Laboratories. Mice(6-10 wks of age) were maintained in pathogen-free conditions and fedrodent chow and water ad libitum. Female Hartley guinea pigs, 4-6 weeksof age, were procured from Charles River Laboratories, and maintained inpathogen-free conditions receiving food and water ad libitum.

Cholera toxin (CT) was purchased from List Biologicals. E. coli heatlabile enterotoxin (LT) was purchased from Swiss Serum and VaccineInstitute (SSVI).

To prepare recombinant CS6 (rCS6), the complete four-gene operon for CS6(approximately 5 kb) was cloned into E. coli strain HB101 (Wolf et al.,1997; Wolf et al., 1997) on a pUC19 derivatized plasmid containing akanamycin resistance gene. rCS6 was produced using this clone in a NewBrunswick BioFlo 3000 fermentor. The fermentation broth was harvested bycentrifugation, and the rCS6 purified by tangential flow filtrationfollowed by precipitation in ammonium sulfate (Wolf et al., 1997). TherCS6 was then buffer exchanged with phosphate buffered saline (PBS).This purified rCS6 was stored at −30° C. until immunization. The purityof rCS6 was determined as >98% by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE), Coomassie blue staining, anddensitometric scanning (Cassels et al., 1992; Schagger & von Jagow,1987).

Clinical ETEC strains E8775 and E9034A were utilized as the sources fornative CS6 (nCS6) and CS3, respectively. Heat and saline extracts of thetwo strains grown on CFA agar (Evans et al., 1977) were treated withammonium sulfate sequentially at 10% saturation intervals (Wolf et a.,1997). The precipitate at 60% and 30% saturation contained the greatestquantity and highest purity of native CS6 and native CS3, respectively,as determined by SDS-PAGE and ELISA assay.

P. falciparum MSP (MSP-1₄₂, 3D7) was expressed in E. coli BL21 (DE3)with a polyhistidine tag (Novagen). Antigen was purified to nearhomogeneity using three chromatographic steps: nickel affinity, Q anionexchange, and CM cation exchange.

Mice were transcutaneously immunized as described previously(Scharton-Kersten et al., 2000). Briefly, the animals were shaved on thedorsum with a No. 40 clipper, which leaves no visible irritation orchanges in the skin, and rested for 48 hr. Mice and guinea pigs wereanesthetized in the hind thigh intramuscularly (IM) or intraperitoneally(IP) with a ketamine/xylazine mixture during the immunization procedureto prevent self-grooming. The exposed skin surface was hydrated withwater-drenched gauze for 5-10 min, and lightly blotted with dry gauzeprior to immunization. Twenty-five to 100 μl of immunizing solution wasplaced on the shaved skin over a 2-cm² area for one hour. The animalswere then extensively washed, tails down, under running tap water forapproximately 30 sec, patted dry, and washed again.

Passive immunization was accomplished by tail vein injection of pooledhyper-immune serum from a matched strain of mice containing an anti-LTIgG titer greater than 10,000 ELISA units. Naïve BALB/c mice wereinjected with 0.5 ml of serum one hour before oral challenge with LT orbicarbonate buffer. Naïve C57BL/6 mice were also passively immunizedusing the same procedure 12 hr before challenge.

An exotoxin challenge model has been described (Richardson et al, 1984).BALB/c mice were fed LT (10 μg in 500 μl) suspended in 10% sodiumbicarbonate (NaHCO₃) solution. C57BL/6 mice were fed LT (100 pg per gramweight in 500 μl of 10% NaHCO₃) based on body weight. Control animalsreceived 500 μl of 10% NaHCO₃ alone. To prevent coprophagy, the micewere transferred to cages with wire mesh flooring. Mice were given 10%glucose water but no food for 12 hr before challenge and duringchallenge. Six hours after the challenge, the animals were weighed andsacrificed. The small intestines were then dissected (pyloric valve toileal-cecal junction), tied off to prevent fluid loss, and weighed.Fluid accumulation was calculated using the formula: FA=(G/(B−G))*1000,where G=gut weight+fluid in grams and B=body weight in grams. Using thisformula, baseline fluid accumulation in untreated or bicarbonate fedanimals is 30 to 150, depending upon the initial body weight of theanimal.

Histopathological studies were performed by Gary M. Zaucha, AVP, ABT,and ACVPM of the Comparative Pathology Division at the Walter Reed ArmyInstitute of Research. Two guinea pigs per treatment group and onecontrol animal were designated for pathology. Animals were euthanized onday two post-exposure for each of the three vaccinations and subject toa complete gross necropsy. Histopathologic examination of the high dosegroup included examination of a full complement of tissues with skin(haired skin and the dorsal lumbar exposure site) and liver evaluated inthe remaining groups. Tissues collected and formalin fixed in the highdose group were brain, pituitary, tongue, lung, trachea, esophagus,thyroid, thymus, heart, pancreas, spleen, liver (with associatedgallbladder), stomach, small intestine, cecum, colon, mesenteric lymphnode, kidney, adrenal, urinary bladder, ovary, uterus, salivary glands,submandibular lymph node, bone marrow (sternum), haired skin, dorsallumbar exposure site, and gross lesions. Histopathologic findings forindividual animals were graded on a scale of 1 to 5 (1=minimal, 2=mild,3=moderate, 4=marked, and 5=severe).

Antibody levels against CT, LT, native CS3, native CS6, rCS6 and MSP-1₄₂were determined by ELISA. Immulon-2 polystyrene plates (DynexLaboratories) were coated with 0.1 μg/well of antigen, incubated at roomtemperature overnight, blocked with a 0.5% casein buffer in PBS, washed,serial dilutions of specimen applied, and the plates incubated for 2 hrat room temperature. IgG (H+L) antibody was detected using HRP-linkedgoat anti-mouse IgG (H+L) (Biorad) for 1 hr. Anti-LT specific IgA levelswere determined as above with HRP-linked goat anti-mouse IgA (Zymed)substituted as the secondary antibody. Secretory IgA (S-IgA) antibodylevels were also measured by ELISA, wherein LT coated plates weresequentially incubated with stool, lung wash, or vaginal wash from naiveand immunized animals, purified rabbit anti-secretory chain (SC)antibody (16-24 hr at 4° C.) (Crottet et al., 1999), andperoxidase-labeled goat anti-rabbit IgG (H+L) (Kirkegaard and Perry) for2 hr at room temperature. Bound antibody was revealed using2,2′-azino-di (3-ethylbenzthiazoline sulphonic acid) substrate (ABTS;Kirkegaard and Perry) and the reaction stopped after 30 min using a 1%SDS solution. Plates were read at 405 nm. Antibody titer results arereported in either OD (405 nm) or ELISA Units, which are defined as theinverse dilution of the sera that yields an optical density (OD) of 1.0.Guinea pig anti-rCS6 ELISAs were conducted as above withperoxidase-conjugated goat anti-guinea pig IgG (Jackson ImmunoResearch)included in the detection step. Anti-SC secondary antibody reacted withantigen coated (rCS6) plates, resulting in high background, whichrendered this assay unsuitable for anti-rCS6 SC detection.

One microliter of rCS6 (0.5 μg), native CS6 (1.6 μg) and native CS3 (0.5μg) were spotted onto nitrocellulose strips (Schleicher and Schuell),and dried overnight. Strips were blocked by incubation in PBS with 0.05%Tween 20 (PBS-T, Sigma) and 1% bovine serum albumin for 2 hr. Primarymouse antibody was diluted to 1:1000 and 1:4000 and incubated with thestrips for 1 hr, followed by three washes of 1, 5, and 10 min, all inPBS-T. Strips were then incubated in goat anti-mouse IgG labeled withhorseradish peroxidase (1:5000 in PBS-T, 30 min). After washing in PBSthree times for 10 min each, the strips were developed with 3,3′diaminobenzidine (DAB, Sigma), hydrogen peroxide (Sigma), and cobaltchloride (Mallinckrodt), as described in Harlow & Lane (1988). Allincubations and washes took place on an orbital shaker at roomtemperature.

Seven days after the last immunization, mononuclear cells were isolatedfrom the spleen and superficial ventral cervical nodes and washed inRPMI 1640 with 50 mg of gentamycin per ml prior to use in the ELISPOTassay as previously described (Hartman et al., 1994; Hartman et al.,1999). Washed spleen and lymph node cells were counted and diluted inculture medium (RPMI 1640 with 2 mM glutamine, 50 mg of gentamycin perml, and 10% fetal bovine serum) to a density of 2.5×10⁶/ml. One hundredml of the cell suspension were inoculated into microwells previouslycoated with 0.1 μg/well of CS6 antigen in carbonate coating buffer, pH9.6, or coating buffer alone. Each sample was assayed in quadruplicate.After incubation at 37° C. for 4 hr, plates were washed and rabbitanti-guinea pig IgG (1:1200), IgA (1:700), or IgM (1:800) (ICNLaboratories) was added. After overnight incubation at 4° C., plateswere washed and alkaline phosphatase-conjugated goat anti-rabbitanti-sera (Sigma) at a dilution of 1:1200 was added. After 2 hr at 37°C., plates were washed and spots were visualized by the addition of 100ml of molten agarose containing 100 mg of 5-bromo-4-chloro-3-indollylphosphate per ml. Spot-forming cells were then counted with astereomicroscope.

Blood contamination was not apparent upon visual inspection of freshlycollected murine stool, lung wash or vaginal wash specimen. Furthertesting with HEMASTIX strips (Bayer) indicated that blood contaminationwas ≦5-20 intact red blood cells per μl or ≦0.015-0.062 mg freehemoglobin per dL.

Stool pellets were collected the day before challenge by spontaneousdefecation, weighed, homogenized in 1 ml of PBS per 100 mg of fecalmaterial, centrifuged, and the supernatant collected and stored at −20°C.

Mice were exsanguinated, the trachea transected, 22-gauge polypropylenetubing inserted, and PBS gently infused to inflate the lungs. Theinfused material was then withdrawn, re-infused for a total of threecycles, and stored at −20° C.

The vaginal cavity was gently lavaged by repeated insertion andaspiration of PBS (80 μl) into the vaginal cavity for a total of threecycles. The vaginal material was spun for 10 min at 3,000 rpm and thesupernatant transferred to a clean container and stored at −20° C.

BALB/c mice were immunized on the skin with MSP1 alone or CT and MSP1 at0, 4, 8 and 12 weeks. Spleen and draining lymph node (inguinal) tissueswere removed 24 weeks after the primary immunization. Single cellsuspensions were prepared from spleen tissue from individual mice orfrom LNs pooled within each group. Cells were cultured at 4×10⁵ per wellin 96-well plates for 5 days at 37° C., 5% CO₂ in the presence orabsence of 10 μg/ml of MSP1 antigen. Concanavalin A (ConA) at 5 μg/mlwas used as a positive control. Culture media contained RPMI 1640(BioWhittaker) supplemented with 10% fetal calf serum (Gibco),penicillin (10 U/ml, BioWhittaker), streptomycin (100 μg/ml,BioWhittaker), L-glutamine (2 mM, Sigma), and Hepes (10 μM, BioRad).[³H]-thymidine (1 μCi/well) was added to the cultures during the last 20hr of the 5-day culture period. Thymidine incorporation was assessed byharvesting cellular DNA onto glass fiber filters, followed by liquidscintillation counting.

CD4⁺T cells were isolated from pooled spleen cells from the CT/MSP1immunization group using a CD4⁺T cell selection column and themanufacturer's instructions (R&D Systems). Cells eluted from the column(CD4⁺) were cultured in 96-well plates at 1×10⁵ cells per well in thepresence or absence of 3×10⁵ irradiated (3000 rad) feeder cells fromnaïve mice. Proliferation assays were conducted in the presence orabsence of antigen stimulation as described above.

Unless otherwise indicated, the ELISA data shown are the geometric meanof values from individual animals, and error bars represent two standarddeviations from the mean. Comparisons between antibody titers and fluidaccumulation (FA) in groups were performed using an unpaired, two-tailedStudent's t test and p values <0.05 regarded as significant.

Immunization on the skin with bacterial adjuvant and the colonizationfactor CS6 results in a protective antibody response. To determine ifTCl would be an effective method for inducing relevant ETEC immuneresponses, mice were immunized four times by TCl with CT and rCS6,assayed for anti-CS6 responses, subsequently challenged orally with CTholotoxin, and the degree of acute intestinal swelling (fluidaccumulation) quantified 6 hr later. A positive control group wasimmunized by the intramuscular route (IM) with 5 μg of rCS6 in alum, anda negative control group received rCS6 alone on the skin. Antibodiesreacting with rCS6 antigen were apparent after the first immunization inanimals receiving either a low (10 μg) or high (100 μg) dose ofadjuvant, and the titers continued to rise after the 1^(st) and 2^(nd)booster immunizations. The immune response to CS6 in the presence ofadjuvant (10 or 100 μg) was greater (p<0.05) than the response seen toantigen alone delivered by TCl at 12 weeks. The geometric mean anti-CS6titers were greatest in the high dose (CT 100 μg) group following thethird immunization, and a higher geometric mean anti-CS6 titer wasproduced using TCl compared to intramuscular immunization, but neitherdifference was statistically significant. Anti-CT titers were elevatedin both of the CT adjuvanted groups at all time points. Animalsimmunized with CS6 alone on the skin failed to develop a consistentantibody response to the antigen or the adjuvant.

Naïve, antigen alone, and CT+CS6 (100 μg/100 μg) on the skin groups wereselected for oral challenge with CT after immunization using TCl. TheCS6 alone and CT/CS6 groups were boosted 11 weeks after the 3^(rd)immunization (19-week study point). Two weeks later, the animals werefed either bicarbonate buffer alone (10% NaHCO₃) or bicarbonate buffercontaining 10 μg of CT, and the resulting intestinal swelling wasdetermined. The intestines from naïve mice fed bicarbonate alone had abaseline fluid accumulation of 103 (range 78 to 146). Oraladministration of CT in naïve mice resulted in a two-fold increase influid accumulation (mean 209; range 164 to 359). Similarly, micevaccinated with rCS6 alone and subsequently fed CT also displayed anapproximate two-fold increase in fluid accumulation (mean 192; range 119to 294). In contrast, mice vaccinated with CT by TCl developednegligible intestinal swelling following challenge (mean 105; range 84to 120), and the fluid response was indistinguishable from that observedin the naïve group fed bicarbonate buffer alone (p<0.5).

Comparable adjuvant effects of CT and LT for transcutaneouslyadministered CS6 antigen. Use of LT as an adjuvant for an ETEC vaccinemay be desirable, as LT is the causative agent in a significant numberof cases of ETEC diarrhea (Wolf et al., 1993), and thus can functionboth as antigen and adjuvant. To test the relative potencies of CT andLT adjuvants for rCS6, mice were immunized on the skin three times at4-week intervals with 100 μg of rCS6 and a range of doses of CT or LT(10, 20, and 100 μg). The resulting serum anti-rCS6 and adjuvant titerswere assessed two weeks after the final immunization. As expected,anti-adjuvant (CT or LT) IgG titers were apparent at all adjuvant doses,and the titers were higher and most consistent among the high (100 μg)dose animals. In contrast, while all of the CS6 and LT/CT groupsdeveloped elevated anti-CS6 titers, the responses were greatest at thelowest LT doses (10 μg vs. 100 μg), and generally comparable to previousexperience by the IM route.

Serum antibodies from mice immunized with rCS6 and LT recognize nativeCS6. Mice immunized with rCS6 produced a high titer of serum IgG thatreacted with the recombinant protein used in the ELISA assay. Whilethese results suggested that TCl might be effective in attenuating ETECinfection and disease, it was important to determine if the inducedantibodies reacted with the native CS6 (nCS6) present on E. coliisolates. To test the specificity of the anti-CS6 response, sera fromrCS6-immunized mice were analyzed for reactivity to native CS6 proteinin ELISA and immunodot blot assays. In the ELISA, each of the threesamples with reactivity to rCS6 and LT demonstrated specificity fornative CS6 protein, but not for native CS3 protein. Similarly, byimmunodot blot assay, mice immunized with rCS6 and LT reacted with boththe immunizing rCS6 antigen and partially purified native CS6, withlittle or no reaction by preimmune sera. None of the mouse sera reactedwith the native CS3 antigen control. Serum from a mouse that wasparenterally immunized (IM injection) with rCS6 responded in a similarway to both native CS6 and rCS6 as did sera from transcutaneouslyimmunized mice. In addition the BALB/c mouse responded in a similarfashion as did the C57BL/6 mice. Thus, TCl using rCS6 induced serumantibody capable of recognizing native antigen.

Antibodies to LT actively and passively protect mice from oral challengewith LT. Although LT, the causative agent in LT-mediated ETEC disease,is very similar to CT and shows cross-protection with cholera toxin Bsubunit (CTB) antibodies (Clemens et al., 1988), direct protectionagainst LT oral challenge using LT antibodies has not been previouslyshown. Mice immunized with LT and rCS6 by TCl were orally challengedwith LT as described. High levels of anti-LT IgG were detected in thesera of immunized mice (geometric mean for BALB/c=36,249 ELISA units;C57BL/6=54,792 ELISA units). For oral toxin challenge, two strains ofmice with different sensitivities to challenge were used. C57BL/6 miceare highly sensitive to the effects of LT toxin challenge compared toBALB/c, and protection in both strains suggests the protective effectmight be observed in more genetically diverse settings. Significantprotection against LT challenge was seen in both strains (p<0.05).

Studies of dog and human ETEC disease suggest that serum antibodycontributes to protection against diarrhea from intact bacteria as wellas isolated toxin (Pierce et al., 1980; Pierce et al., 1972; Pierce &Reynolds, 1974). Consistent with this premise, we, and others, havepreviously reported that a transcutaneously elicited serum factorprotects animals from a lethal intranasal challenge with CT (Beignon etal., 2001; Glenn et al., 1998b). Thus, we postulated that a serumfactor, presumed to be antitoxin antibody, might also contribute to theprevention of toxin-induced intestinal swelling in transcutaneouslyimmunized mice. The host-protective role of antitoxin antibody serum wasevaluated by quantitating the intestinal swelling elicited by oral toxinchallenge of naïve and passively immunized mice that received serum fromanimals which were treated by TCl. The effect of passive immunizationwas evaluated in both BALB/c and C57BL/6 mouse strains. Oraladministration of LT to naïve mice consistently induced fluidaccumulation that was apparent upon visual inspection. In contrast,passively immunized mice developed negligible fluid accumulation of amagnitude comparable to that observed in the groups fed buffer alone.Thus, the passively immunized mice given antibody from transcutaneouslyimmunized mice were protected from the sequelae of oral toxin challenge.Together, these results indicate that transcutaneously immunized miceproduce serum antibodies capable of protecting animals from toxinexposure.

Mucosal IgG, IgA and secretory IgA responses specific for ETEC antigenfollowing TCl. While serum IgG responses are associated with hostprotection against many infectious agents, mucosal immune responses areconsidered to be important for attenuation and prevention of mucosallyacquired pathogens, particularly intestinal pathogens, such as ETEC. Todetermine if TCl with rCS6 induces mucosally detectable antibodyresponses, IgG and S-IgA responses were analyzed in fecal, lung andvaginal specimens harvested from mice immunized on the skin with rCS6and adjuvant. C57BL/6 mice were immunized three times with CS6 alone,LT/CS6, or CT/CS6. CS6-specific IgG was evaluated in fecal, lung andvaginal wash specimen collected nine weeks after the 3^(rd)immunization. Immunization with rCS6 alone failed to induce elevatedCS6-specific IgG in either fecal, lung or vaginal wash specimen. Incontrast, 3 of 3 animals in the CT/CS6 group contained detectableanti-CS6 IgG in both lung and vaginal wash specimens. Similarly, CS6specific IgG antibody was observed in lung and vaginal specimen from theLT adjuvanted group and in fecal specimen from CT and LT adjuvantedgroups, although the responses were less consistent. The method ofcollection with fecal samples may have hampered the consistency of fecalantibody results, especially if the responses were modest, and othercollection methods are being investigated.

Locally produced IgA is typically a dimeric protein associated withsecretory chain (SC) that allows transport across the epithelialmembranes. To determine if TCl could induce secretory IgA (S-IgA)production, animals were immunized twice on the skin with LT and theantigen-specific IgG, IgA and S-IgA titers evaluated in mucosal specimenby ELISA. As compared to specimen from naïve animals, immunization withLT induced antigen-specific IgG and IgA in the fecal and vaginalspecimen of 10 of 10 immunized mice. More importantly, S-IgA was readilydetected in all 10 fecal and vaginal specimens tested.

Induction of protective antitoxin immunity following co-administrationof CT and a malarial vaccine antigen. Targeted vaccination againstmultiple infectious agents is desirable in developing countries whererelatively low life expectancies and high morbidity and mortality ratesare associated with infection of individuals with more than onepathogen. To determine whether TCl might be employed for inducingprotection against multiple unrelated infectious agents, mice weresimultaneously vaccinated with CT and a C-terminal 42 kDa fragment of aPlasmodium falciparum protein, merozoite surface protein 1 (MSP-1₄₂). CT(0, 10 or 100 μg) and MSP (100 μg) proteins were applied to the skin at0, 4, 8, and 13 weeks. Mice were considered responsive to MSP if thepost-immunization titer was 3-fold the OD measured in thepre-immunization serum at a 1:100 dilution. Based on this criterion, MSPantibodies were detected in serum from mice immunized with CT and MSPtogether but not in serum collected from the control group (MSP alone)nor that harvested prior to immunization (prebleed). To evaluate theeffectiveness of the anti-CT antibody response in the dual immunizedmice, animals from the CT (100 μg) plus MSP (100 μg) group thatdeveloped high levels of anti-CT antibodies were orally challenged withCT and the degree of intestinal swelling (fluid accumulation) wascompared with that induced in mice vaccinated with MSP alone. All of theanimals immunized on the skin with CT and MSP together exhibited lowerfluid accumulation levels (p<0.01) than comparably challenged mice inthe MSP alone exposure group. Moreover, spleen and draining lymph nodecells from the immunized mice exhibited a strong antigen-specificproliferative response in vitro in the draining lymph node and spleen,to which CD4⁺T cells contributed. These results suggest that theantibodies to the adjuvant may confer protection against LT-mediateddisease while functioning as an adjuvant for other antigens, such ascandidate malaria vaccine antigens.

Immune responses to ETEC antigens in guinea pigs. To assess thecapability for induction of antibody secreting cells (ASC) by TCl, anestablished guinea pig ASC animal model was selected. The guinea pig isa conventional model for assessment of toxic reactions in response toepicutaneous administration of adjuvant and antigen. Guinea pigs wereexposed to increasing doses of LT (12 to 100 μg) and rCS6 (25 to 200 μg)on the skin on days 0, 21 and 42. Serum was collected for serology ondays 1, 20, 41 and 56, and antibody titers to the antigen and adjuvantdetermined by ELISA. Similar to the results of the mouse studies, TCladministration of the rCS6 and LT vaccine resulted in induction of CS6and LT antibody responses that appeared to be dose related with respectto CS6 and LT concentration (Table 2). The finding of serum antibody toCS6 was confirmed by the observation of ASC specific for CS6 in spleenand draining lymph node tissues. ELISPOT assay conducted on freshlyisolated cells from sham PBS and CS6/LT immunized animals revealed asignificant elevation (p<0.05) in the number of rCS6-specific IgGproducing cells in 4 of 4 of the antigen exposed animals.Antigen-specific IgA and IgM producing cells also seemed enhancedalthough the actual number of cells detected was smaller and lessconsistent (Table 3).

TABLE 2 Serum anti-CS6 and anti-LT IgG in guinea pigs after TCI with CS6and LT Adjuvant/Antigen Mean serum IgG (ELISA Units) per mouse (μg)Pre-bleed 3 wk 6 wk 8 wk LT(100)/CS6 (200) n = 7 n = 7 n = 5 n = 3Anti-CS6 IgG 30 55 1588  4383 Anti-LT IgG 57 104  1258  3764 LT(50)/CS6(100)  n = 10  n = 10 n = 8 n = 1 Anti-CS6 IgG 23 55 1155  12471 Anti-LT IgG 52 87 297 4933 LT(25)/CS6 (50) n = 6 n = 6 n = 4 n = 2Anti-CS6 IgG 24 26 103 1084 Anti-LT IgG 43 77 106  381 LT(12)/CS6 (25) n= 6 n = 6 n = 4 n = 2 Anti-CS6 IgG 13 26  68  243 Anti-LT IgG 35 65 112 153 PBS n = 6 n = 6 n = 5 n = 2 Anti-CS6 IgG 18 27  54  36 Anti-LT IgG42 73 113  95

Two guinea pigs were designated for pathology per treatment group andone from the control group at each of the three exposures (Table 2). Afull complement of tissues was subject to histopathologic evaluation inthe high dose group. Tissues collected from the remaining groups(control, low, and mid range) were limited to the skin and liver. Grossnecropsy and histopathology of the high dose group failed to demonstratesystemic lesions that could be attributed to the administration of thetest article at any of the exposures. Hepatic necrosis was observedgrossly in all animals including the PBS controls, but there was nocorrelation of the finding with the treatment groups. Serum alanineaminotransferase, aspartate aminotransferase, alkaline phosphatase,sodium, potassium, and blood urea nitrogen were evaluated and determinedto be normal in all treatment groups.

TCl with LT/CS6 resulted in minimal to mild inflammatory changes limitedto the local site of exposure and increases in the dose above 25 μgLT/50 μg CS6 had no appreciable effect on the severity of the localresponse. Typical findings were infiltration of the superficial dermisby low numbers of granulocytes and lymphocytes (inflammation), mildthickening of the epidermis by hyperplasia of keratinocytes(acanthosis), and occasional small foci where epidermal cells had lostcohesion, resulting in the formation of intraepidermal vesiclescontaining free keratinocytes (acantholysis). Minimal changes were seenin the lowest dose group (LT 12 μg/CS6 125 μg) at the three time points,and no skin changes were observed in the PBS exposed controls. In bothmice and guinea pigs, there was no clinical progression in the severityof the skin findings with repeated immunization, and where seen, thevesicles either resolved or crusted and resolved spontaneously overseveral days.

TABLE 3 Individual guinea pig anti-CS6 ASC and IgG antibodies induced byTCI ASC per Immunization Million Cells Group Animal IgG IgA IgM PBS 1 11 2 2 2 0 4 CS6/LT 1 8 1 12 2 42 3 1 3 31 1 4 4 63 5 7

HUMAN EXAMPLES

Healthy male and female volunteers, aged 18 to 45 years were recruitedfrom the Washington, D.C. metropolitan area. Exclusion criteria includedtravel to an ETEC-endemic area in the previous year, recent history oftraveler's diarrhea, pregnancy, infection with HIV, hepatitis B virus,hepatitis C virus, and allergy to antibiotics.

The vaccine components consisted of CS6 antigen mixed with LT. CS6 wasproduced under current good manufacturing practices (GMP) at the ForestGlen Pilot BioProduction Facility of the Walter Reed Army Institute ofResearch. The bacterial strain used for the production of CS6 wasconstructed from E. coli strain HB101 and a plasmid containing thefour-gene operon necessary for CS6 expression inserted by recombinanttechniques. The CS6 genes were cloned from ETEC strain E8875 (Wolf etal., 1997). The major steps in the production of CS6 included: bacterialfermentation; purification of the CS6 from the fermentation broth bytangential flow filtration followed by ammonium sulfate precipitation;intermediate storage of the bulk CS6 protein in phosphate bufferedsaline (PBS) solution at −80° C.; thawing, stirring, and distributioninto vials; and storage at −80° C. CS6 was formulated as purifiedprotein in 2 ml serum vials with gray split rubber stoppers sealed withaluminum crimps. Each vial contained 0.9 ml of (1.3 mg/ml) CS6 proteinin PBS. Native LT of E. coli was produced under current GMP at the SwissSerum and Vaccine Institute (SSVI). The LT was produced from E. colistrain HE22 TP 235 Km. The LT was supplied as lyophilized powder. Eachvial contained 500 μg of lyophilized LT, and was reconstituted with 1 mlsterile water. The doses of adjuvant (LT) and antigen (CS6) byvaccination group are shown in Table 4.

The vaccine was administered in three doses. The first dose wasadministered on day 0, and the second and third immunizations on days 28and 84 respectively after the first immunization. The vaccine wasadministered transcutaneously using a semi-occlusive patch consisting ofa 2×2 inch cotton gauze matrix (two-ply Kendall #2556) with a 2×2 inchpolyethylene (SARAN WRAP) backing covered by a 4×4 inch TEGARDERMdressing (semi-occlusive, 3M cat # 1616).

At the time of vaccination the vaccine was applied in 500 μl of sterilesaline and administered as a split dose on each upper arm. Each splitdose contained the corresponding dose of CS6 (antigen) alone or incombination with 250 μg of LT (adjuvant). The upper arm was positionedin a half-extended manner on an examination table and prepared by gentlyrubbing five times with an isopropyl alcohol (70%) swab. The cottongauze was placed on each upper arm and the immunization solution wasapplied to the gauze with a syringe. The polyethylene backing was thenplaced over the impregnated cotton gauze and covered with the Tegadermdressing. Volunteers remained in the research clinic for 20 minfollowing patch application for observation. Volunteers were instructednot to touch the patches or engage in strenuous physical activity duringthe time the patches were worm. The patches were removed 6 hr afterapplication (range: 4-8 hr). The immunization sites were then rinsedwith 500 ml of water and patted dry. The volunteers were instructed tobathe or shower in the evening but to refrain from heavy scrubbing ofthe immunization site with soap to avoid unusual irritation of the skin.Volunteers were re-immunized at 28 and 84 days after the firstimmunization. Each volunteer received the same dose of vaccine on eachimmunization.

Volunteers were observed for 20 min after each dose for occurrence ofimmediate adverse effects. The volunteers were given a diary to recordsigns and symptoms observed after vaccination. Reported symptoms weregraded as mild (noticeable), moderate (affecting normal dailyactivities), or severe (suspending normal daily activities). Thevolunteers were evaluated at 24 hr and 48 hr for clinical assessment andevaluation of possible side effects. Volunteers who showed signs ofvaccine skin reactions were instructed to return to the clinic at 72 hrfor additional clinical assessment. Volunteers were then followed asneeded until side effects had completely resolved. One of the volunteerswho developed a skin rash in the site of immunization was asked toundergo a skin biopsy. This biopsy was performed by a dermatologist,following standard procedure, and under a separate written informedconsent.

Antibody-secreting cells (ASC) immune responses to the vaccine antigenswere chosen as an immunological endpoint for this study, since previousstudies have shown that ASC responses correlate with mucosal intestinalimmune responses (Wenneras et al, 1992). Venous blood samples wereobtained from the volunteers on day 0 before immunization, and on days7, 28, 35, 56, 84, 91, 98, and 112 after the first immunization. Bloodspecimens were collected using the VACUTAINER system of EDTA-treatedtubes (Becton Dickinson). Peripheral blood mononuclear cells (PBMC) wereisolated from the blood sample by gradient centrifugation onFicoll-Hypaque (Sigma) and were assayed for total and vaccine-specificnumbers of IgA and IgG ASC by the ELISpot technique (Czerkinsky et al.,1988; Wenneras et al, 1992). Individual wells of nitrocellulose-bottomed96-well plates (Millititer H A; Millopore, Bedford, Mass.) were coatedwith 0.1 ml of purified CS6 (20 μg/ml) or GM₁ ganglioside (0.5 μg/ml)and incubated overnight at 4° C. After a PBS wash, GM1-coated wells wereexposed to LT (0.5 μg/ml) for 2 hr at 37° C. After being washed withPBS, the plates were blocked with complete RPMI medium (Gibco)supplemented with 5% fetal calf serum (Gibco) and 50 μg/ml gentamicin(Gibco). The PBMC were adjusted to 2×10⁷ viable cells/ml in completeRPMI medium. A final 0.1 ml suspension of PBMC was added to each well(1×10⁶ PBMC added per well), and plates were incubated for 4 hr at 37°C. in 5.0% CO₂. Plates were washed, incubated overnight at 4° C. with amixture of two affinity-purified goat anti-human immunoglobulinantibodies with distinct isotype specificities, one conjugated toalkaline phosphatase (IgG) and the other conjugated to horseradishperoxidase (IgA) (Southern Biotech Associates) and exposed to theappropriate chromogen-enzyme substrate (Sigma). Spots, corresponding toa zone of antibodies secreted by individual cells, were enumerated intriplicate wells under 40× magnification, with data expressed as thenumber of spot-forming cells per 10⁶ PBMC.

As previously described (Ahren et al., 1998; Jertborn et al., 1998;Jertborn et al., 2001), we defined a positive ASC response as a ≧2-foldincrease over baseline value of the ASCs per 10⁶ PBMC, when the numberof ASCs was ≧0.5 per 10⁶ PBMC in the baseline sample. If the number ofpreimmune ASCs was less than 0.5 per 10⁶ PBMC, a value of >1.0 per 10⁶PBMC after dosing was considered a positive response.

Venous blood samples were obtained from the volunteers beforeimmunization and on days 14 and 28 after each immunization formeasurements of serum antibody titers. IgA and IgG antibody titersagainst LT were measured by the GM1-ELISA method (Jertborn et al., 1998;Svennerholm et al., 1983), and those against the CS6 were determined byELISA methods as previously described (Hall et al., 2001; Stoll et al.,1986). LT (provided by SSVI) and CS6 (GMP Lot 0695, WRAIR) were used assolid-phase antigens. The LT and CS6 used for the ELISA essays were fromthe same lots used for the vaccine preparation. Individual microtiterwells (Nunc-Immunoplates) were coated with GM1 ganglioside (0.5 μg/ml)(Sigma) at room temperature overnight, or with 0.1 ml of a 1.0 μg/mlpreparation of CS6 at 37° C. overnight. GM1-coated wells were thenwashed with PBS and incubated with 0.1 ml of LT (0.5 μg/ml) for 2 hr at37° C. After blocking with 0.1% bovine serum albumin (Sigma), the serumsamples were threefold serially diluted (initial dilution 1:5) and thenincubated at room temperature for 90 min. Bound antibodies weredemonstrated by addition of rabbit anti-human IgA or IgG conjugated withhorseradish peroxidase (Jackson ImmunoResearch Laboratories, PA) andincubated at room temperature for 90 min, followed by addition ofO-phenylenediamine (OPD)-H₂O₂ (Sigma). The endpoint titers were assignedas the interpolated dilutions of the samples giving an OD of 450 nm of0.4 above background (absorbance at 450 nm). Titers were adjusted inrelation to a reference specimen included in each test to compensate forday-to-day variation. For both antigens, pre- and post-dosing serumsamples from the same volunteer were always tested side by side. Theantibody titer ascribed to each sample represented the geometric mean ofduplicate determinations performed on different days. Reciprocalendpoint titers <5 were assigned a value of 2.5 for computationalpurposes. Based on our calculations of the methodological error of eachELISA, previous to the study, we defined a significant response(seroconversion) as ≧two-fold increase in endpoint titer between pre-and post-immunization specimens (Jertborn et al., 1986), with the addedcriterion that the post-immunization reciprocal titer be ≧10.

All volunteers receiving the three scheduled doses of vaccine wereincluded in the post-dosing safety and immunogenicity analyses.Proportions were compared using the 2×nχ² test at α=0.05, power=0.80.The Fisher's exact test was used in 2×2 tables when the number containedin one of the cells was less than 5 (Sahai & Khurshid, 1995). The mediannumber of ASC and median plasma antibody titer fold increases werecompared separately using the Wilcoxon rank test to assess the boostingeffect of each consecutive dose of vaccination (Forrester & Ury, 1969).All statistical tests were two-tailed.

Informed consent was obtained from all volunteers, and the human useguidelines of the U.S. Department of Defense were followed in theconduct of this trial. Thirty-three volunteers were enrolled andreceived at least one dose of the study vaccine. The protocol wasapproved by the Institutional Review Board of the Office of The SurgeonGeneral, U.S. Army. The volunteers were 21 to 44 years of age; 17females and 16 males; 15 black, 16 white, and two Asian. Of the 33volunteers, seven did not complete the study for reasons unrelated tothe study: conflict with their work schedule (4), moving from the D.C.metropolitan area (2), and admission to a local clinic for illegal druguse (1). Twenty-six volunteers received the three scheduled doses of thevaccine and completed all the post-vaccination follow-up visits, and thedata on these volunteers is shown. These volunteers were 21 to 44 yearsof age; 13 females and 13 males; 12 black, 12 white, and two Asian. Thenumber of volunteers by vaccine dose is shown in Table 4.

TABLE 4 Number of volunteers by vaccine group (n = 26) Dose of Adjuvant(LT)* Dose of antigen (CS6)* 500 μg 0 μg  250 μg 5 2  500 μg 5 1 1000 μg5 1 2000 μg 4 3 Total 19  7 *Dose was split between two patches

Of the volunteers receiving a combination of LT/CS6, 74% (14/19)developed a maculo-papular rash at the site of vaccination. Novolunteers receiving CS6 alone developed a rash. The reaction was mildin 13 volunteers and moderate in one volunteer. White volunteersdeveloped the rash significantly more frequently than black volunteers(11/11 vs. 3/8, p<0.005). Seven reactions occurred after theadministration of the 2nd dose and 14 occurred after the third dose; all7 volunteers with a 2nd dose-related rash also developed the rash afterthe application of the 3rd dose. The clinical diagnosis was contactdermatitis (delayed type hypersensitivity-DTH). One volunteer with thecharacteristic rash underwent a biopsy of the affected skin afterreceiving the third dose of LT/CS6. The biopsy showed mild dermalchronic inflammation (lymphocytic) with focal spongiosis. Thepathological diagnosis was sub acute spongiotic dermatitis,characteristic of DTH. The rash usually began within 24 hrs after patchapplication. Rash developing after the second dose lasted a median of 9days, (range 1-14); the third dose rash lasted a median of 6 days,(range 1-11). Volunteers were offered 0.1% triamcinolone cream forrelief of potential vaccine related symptoms. None of the subjects usedthe cream after the first or second immunizations. Eight patients withrashes that occurred after the third dose were treated with thetriamcinolone cream. There were no apparent clinical differencesregarding the appearance and severity of local symptoms (pruritus) orsigns (erythema, papules) when compared by vaccination dose. There wereno statistically significant differences in the magnitude of theserological immune responses between the users of triamcinolone cream ascompared to non-users.

Immune responses were detected only in volunteers receiving bothadjuvant and antigen, although one volunteer who received only two dosesof CS6 alone had a positive anti-CS6 IgA response (but no CS6 IgG) at asingle time point. There were no significant differences in thefrequency or the magnitude of the serum antibody or ASC responses to LTand CS6 between the four groups that received the adjuvant and antigencombination; therefore, data were pooled for further statisticalanalysis and presentation. All volunteers (100%) receiving LTdemonstrated a serum anti-LT IgG response, and 90% produced anti-LT IgA.Anti-CS6 serum antibody responses rates were lower than the anti-LTresponse rate with 68% and 53% of volunteers showing greater thantwo-fold rise in anti-CS6 IgG and IgA titers, respectively. Theindividual peak fold rises in serum antibodies to LT and CS6 aredepicted in FIG. 1. Robust responses to both LT and CS6 were observedwith serum antibodies, although there was a great deal of variability inthe magnitude of the response. The mean anti-LT IgG response to LTexceeded the mean fold response previously described by nearly a log(Glenn et al., 2000), and was greater than the response to CS6.

The kinetics of the serum antibody responses are depicted in FIG. 2. Thepost-dose serum antibody titers for each group were combined and arepresented as geometric mean titers with 95% confidence intervals. Thekinetics of the immune responses to LT differ markedly from the kineticsof the response to CS6 in that strong priming and boosting responses toLT were seen whereas the CS6 responses were primarily seen withboosting. Memory responses to CS6 appear to occur, as suggested by thesignificant difference in the pooled anti-CS6 IgG and IgA responsesafter both the second and third immunization.

The percent of ASC response rate and median number of antigen-specificASC×10⁶ PBMC are shown in Table 5. Both CS6 and LT-specific ASC weredetected. The time and magnitude of peak number ASC for each individualresponder by specific ASC type are depicted in FIG. 3. In the majorityof responders, peak ASC were detected after the second or thirdimmunization. All seven volunteers that demonstrated anti-CS6 IgG ASChad their peak number of ASC after the third immunization.

TABLE 5 Antigen-specific ASC responses (n = 19) Number of Median NumberASC ASC type responders (%)* per 10⁶ PBMC (range)** Anti-LT IgG 15 (79)   9 (1.3-49) Anti-LT IgA 7 (37) 2.4 (1.3-46) Anti-CS6 IgG 7 (37)   6(1.7-77) Anti-CS6 IgA 8 (42) 2.4 (1.7-12) *A positive ASC response wasdefined as a ≧2-fold increase over baseline value of the ASCs per 10⁶PBMC, when the number of ASCs was ≧0.5 per 10⁶ PBMC in the baselinesample. If the number of preimmune ASCs was less than 0.5 per 10⁶ PBMC,a value of >1.0 per 10⁶ PBMC after dosing was considered a positiveresponse. **Only positive responses were included when calculating themedian number of ASC (range).

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causesof childhood diarrhea in developing countries. It is also the principalcause of traveler's diarrhea. Clinical manifestations of disease arecaused by the bacteria secreting one or two enterotoxins, heat-labileenterotoxin (LT) and the poorly immunogenic heat-stable enterotoxin(ST), both interact with the intestine to cause watery diarrheacharacteristic of the disease. A requirement for infection is theability of ETEC organism to adhere to the intestinal epithelium. Thisoccurs through structures on the outer membrane called fimbrie. As aclass, these structures are antigenically distinct and namedcolonization factor antigens (CFA). To date, 20 different colonizationfactors (CF) have been identified. The most prevalent, and relevant tohuman disease, include CFA/I, CFA/II and CFA/IV. CFA/II is composed ofthree separate antigens named as coli surface antigen 1 (CS1), CS2 andCS3. CFA/IV is composed of three antigens, CS4, CS5 and CS6.

The age-associated decline in the incidence of ETEC infections in thedeveloping world has been attributed to the development of protectiveimmunity. Epidemiology studies demonstrate that infants infected becomeresistant (protected) from reinfection with the same strain. It wasshown that volunteers experimentally infected are protected fromrechallenge with a homologous strain. Human volunteers challenged with aheterologous ETEC strain are not protected against clinical disease.

The CFA's have been identified as likely candidates for evaluation ofETEC vaccines. However, for broad range coverage against naturalinfection, an ETEC vaccine must consist of multiple colonization factorantigens. The broadest range of coverage (80% -95%) requires thedevelopment of a multivalent vaccine consisting of several CFA (CS3, CS6and CFA/I) and enterotoxin (LT and ST). Although oral administration ispossible, these protein antigens are sensitive to hydrolysis at low pHand enzymatic degradation in the stomach. In addition, large doses ofthe vaccine are required for oral vaccination are not practical as aproduct. ETEC vaccine is not amenable to other routes of administrationsince the enterotoxins (LT and ST) are reactogenic (diarrhea) andinflammatory when administered by other routes, including oral, nasal,pulmonary, rectal and parenteral.

The efficiency and safety of TCl has been applied to this vaccinationproblem. In the following examples, it is demonstrated that multivalentcombinations of CS3, CS6, CFA/I, LT and ST can be effectively deliveredthrough the stratum corneum with or without penetration enhancement andthey induce an immune response against each component of the multivalentvaccine. Vaccination by TCl requires a low dose of the antigens (CS3,CS6 and CFA/I) and the immune response is significantly augmented bysimultaneous co-administration of an adjuvant. TCl can be preformedsafely and without eliciting serious, adverse side effects. We describeantigen and adjuvant doses that are effective, dosing regimens, methodsfor optimizing delivery of the vaccine into the skin to antigenpresenting cells, and formulations which are stabilizing andpharmaceutically acceptable for transcutaneous immunization.

Materials and Methods

Preclinical studies were conducted to establish the optimal method forTCl with a complex mixture of CFA and LT. The objective of the followingstudy was to demonstrate the feasibility of transcutaneous vaccinationwith mixtures of CS3, CS6, CFA/I, LT and STa. In these studies, adultC57BL/6 mice (7-8 weeks old) were used.

Mice were shaved on the dorsal, ventral surface at the base of the tail(24-48 hrours) prior to vaccination. All mice were anesthetized byintraperitoneal injection of 25 μl of a mixture of ketamine (100 mg/ml)and xylazine (100 mg/ml). The shaved site was pretreated by hydrationwith saline or a mixture of 10% glycerol and 70% isopropyl alcohol.While still fully hydrated, the skin was gently treated by one of twomethods to disrupt the outermost layer of skin, the stratum corneum(SC). Tape stripping was preformed by applying 3 M or D-squame® adhesivetape to the prepared surface followed by gentle removal of the tape 10times. Alternatively, the hydrated skin was pretreated by mild abrasionwith emery paper (GE Medical Systems) or a swab containing pumice(PDI/NicePak). The skin surface was gently buffed 10 times using anup-and-down motion. Immediately prior to application, a Nu-gauze pad (˜1cm²), affixed on an adhesive backing, was loaded with 25 μl volumecontaining different combinations of CS3 (25 μg), CS6 (25 μg), CFA/I (25μg), STa (8 μg) and LTR192G (25 μg). The vaccine-loaded patches wereapplied overnight (˜18 hr), removed, and the skin rinsed with water. Allmice received two or three doses two weeks apart on day 0, 14 and 28.

Peripheral blood was obtained by lacerating the tail vein. The blood wascollected in a tube, allowed to clot, centrifuged, and the serumcollected. Serum samples were collected on day 0 (pre-immune), day 14,day 28 and day 42 (two weeks after the third dose). The serum was frozenat −20° C. until evaluated for antibodies to the vaccinating antigens(CS3, CS6, CFA/I and STa) and adjuvant (LTR192G). The latter wasprovided by the U.S. Navy Medical Research Center.

Fecal samples were collected on day 35 (7 days after the thirdimmunization). Fresh samples were collected from each mouse andextracted with PMSF (3 μg/ml in saline). The samples were agitated(VORTEX mixer) to form a suspension and clarified by centrifugation(3,000 rpm, MICROFUGE). The clarified supernatants were recovered andstored at −20° C. until evaluated with an ELISA method for mucosal IgGand IgA.

An enzyme linked immunosorbent assay (ELISA) method was used to assessthe serum IgG. Ninety-six well plates were coated with 1 μg antigen/100μl per well overnight at 4° C. After washing with phosphate bufferedsaline and Tween 20 (PBS-T), the plates were blocked with 100 μl ofblocking buffer (0.5% casein and 0.5% bovine serum albumin) for 1 hr atroom temperature. After washing the plates with PBS-T, the samples wereserially diluted (serum samples 3-fold serial dilution and fecal samples2-fold serial dilution). The plates were incubated overnight at 4° C.The plates were washed with PT buffer and 100 μl of optimally diluted(1:2,000) goat anti-mouse IgG conjugated with HRP (Bio Rad) or HRPconjugated goat anti-mouse IgA (Zymed) was added to each well. Theplates were incubated for 2 hr at room temperature, washed with PTbuffer and 100 μl of substrate ABTS (KPL) was added to the wells and thereaction allowed to develop for 30 min. The reaction was stopped byadding 100 μl of 1% SDS solution (Gibco). The optical density was readat 405 nm with an ELISA plate reader and the data analyzed using SoftmaxPro 2.4 software (Molecular Devices).

Serum antibodies to formalin inactivated ETEC whole cells weredetermined by the ELISA method. Enterotoxigenic E. coli strain E243778were cultured in on agar plates, the cells harvested and inactivated in2.5% formalin overnight at room temperature. The wells were coated with5×10⁵ killed whole cells (EWC). The plates were prepared as describedfor other antigens. Serum antibodies to EWC was determined by the methoddescribed above.

Mice were sacrificed by asphyxiating with carbon dioxide and the spleenand inguinal lymph nodes removed. The tissues were maintained on ice intubes containing RPMI 1640 medium (Gibco). Single cell suspensions wereprepared by grinding the tissue with the barrel of a 5 cc syringe.Tissue debris was allowed to settle to the bottom of the tube and thecell suspension was transferred to another tube. The cells were washedtwice with RPMI 1640 medium and suspended in culture medium (RPMI 1640,10% FBS, 2 mmol of L-glutamine and 2 mmol pen-strep).

Ninety-six well filtration plates (Millipore Bedford, Mass.) were coatedwith 100 μl of 3 μg/ml antigens in PBS and incubated overnight at 4° C.The plates were rinsed three times with PBS, blocked with 2% BSA (Sigma)for 1 hr and rinsed with PBS. Cells were dispensed at 100 μl per well inculture medium (RPMI 1640, 10% FBS, 2 mmol of L-glutamine and 2 mmolpen-strep) and plates were incubated overnight at 37° C. in a humidified5% CO₂ incubator. The plates were washed four times with PBS-0.05% Tween20 (PBS-T). The cells were lysed by hypotonic shock with water. Onehundred μl of biotin conjugated goat anti-mouse IgA (SouthernBiotechnology) or biotin conjugated goat anti-mouse IgG (Amershan)1:2000 dilution in 2% BSA in PBS. The plates were incubated at roomtemperature for 2 hr. After the plates were washed with PBS-T, 100 μl ofalkaline phosphatase-labeled avid in D antibody (Vector) 1:2000 wereadded to each well and incubated for an additional 2 hr at roomtemperature. The plates were washed with PBS-T and 100 μl of BCIP/NBTsolution (Kirkegaard & Perry) was added to the wells and the platesincubated 5 to 30 min at room temperature until blue spots develop. Theplates were washed with distilled water to stop the reaction. Antigenspecific ASC were visualized as blue spots, which were counted with adissecting microscope and recorded as IgG-ASC or IGA-ASC per 10⁶ cells.

In Vitro Assay for Characterizing Neutralizing Antibodies to the ETECEnterotoxin LT

Heat-labile enterotoxins from E. coli (LT) is produced as a multisubunittoxins with A and B subunits. After the initial interaction of theenterotoxin with the host cell membrane receptor (GM1 ganglioside), theB subunit facilitates the penetration of the A subunit through the cellmembrane and into the eukaryotic cell. With chemical reduction, this Asubunit dissociates into two smaller peptides: A₁ catalyzes theADP-ribosylation of the stimulatory GTP-binding protein in the adenylatecyclase enzyme complex on the basolateral surface of the epithelialcells. This results in increasing the intracellular level of cyclic AMP(cAMP). The increase in cAMP causes secretion of water and electrolytesinto the small intestine resulting in clinical disease.

In cell culture, LT binds with high affinity (K_(D)=7.3×10⁻¹⁰) to theGM1 gangiloside receptor, which is expressed by many eukaryotic tissuesand cells. LT causes striking morphologic changes to many eukaryoticcells (for example, CHO and Y1) in cell culture. Using this property, anin vitro assay method was developed to determine if antibodies elicitedthrough TCl inhibit LT (neutralize) binding to the GM1 gangliosidereceptor. In these studies, CHO cell were cultured in F12 mediumsupplemented with 10% fetal calf serum (FCS). The cells were maintainedin log phase growth at 37° C. in 5% CO₂. The cells were trypsinized fromT flasks and plated into 96 well plates at 5×10³ in 0.2 ml of F12supplemented with 1% FCS. Pre-immune (day 0) and post-immune (day 321)sera were collected from volunteers that had been transcutaneouslyvaccinated with LT and determined by the ELISA method to have antibodiesto LT. These sera were diluted 1:4 with culture media and 2-foldserially diluted up to 1:8192. An equal volume of diluted serum wasmixed with LT for 1 hr at 37° C. Fifty μl of serum/LT (containing 6.5 ngLT) was transferred to duplicate freshly plated CHO cells cultures(5×10³ cells in 150 μl medium). The cells were cultured at 37° C. for 24hr. At the end of the culture period, the media was removed, the cellswashed with F12, fixed with methanol and stained with Giemsa stain. Thecultures were examined with a inverted light microscope and culturesgraded for normal appearance or elongated morphology. The results wereexpressed as the lowest dilution of serum that blocked (neutralized)cell elongation by greater than 90% within the culture.

Methods for Conjugating Heat Stable Toxin (STa) to LT or Other CarrierProtein to Improve Immunogenicity and Transcutaneous Delivery

STa (lot 1184A, List Biological) conjugation to LT involves two steps.The first step was to maleimide activate LT, 400 μg of LT (lot 200100,Berna Biotech) was dissolved in 400 μl of 0.1 M phosphate buffer 0.15 MNaCl buffer (pH 7.2). 160 μl ofsuccinmidy1-6[((β-maleimidopropipropionamido0 hexanoate] (SMPH, Pierce)about 8 μl to LT solution and incubated for 90 min at room temperature.The reaction was desalted on a desalting column (Pierce) by using PBSand fractions were collected. The activated LT peak was pooled andprotein determined by a BCA assay (Pierce). The second step isconjugation; 80 μg of STa was mixed with the maleimide activated LT andincubated overnight at 4° C. The ST-LT conjugate was dialyzed against500 ml PBS buffer.

Purified STa (100 μg) was coupled to 800 μg of chicken egg ovalbumin(OVA, Sigma) in 1 ml of reaction mixture containing 10 mg of1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (Pierce) and 0.1 Msodium phosphate buffer (pH 5.5). The reaction mixture was dialyzedagainst phosphate buffer saline (20 nm, pH 7.2, PBS) for 4 hr.

Conjugation was confirmed by shift in the molecular weight of ovalbuminand LT-B using SDS-PAGE method. Each sample was dissolved in 4× samplebuffer (Invitrogen) and heated 100° C. for 5 min and analyzed the NuPAGE4-12% Bis-Tris gel (Invitrogen). After electrophoresis, protein bandswere visualized using a silver staining kit (Invitrogen) and molecularweights of the conjugates were determined relative to internal referencestandards run on the gels.

Example 1 Comparison of the Serum IgG Response to Transcutaneous andIntradermal Vaccination with ETEC Colonization Factors

The purpose of this study was to compare the immune response elicited bytranscutaneous vaccination, to that of intradermal, vaccination with CS3and CS6. Groups of mice were pretreated by tape stripping 10 times toremove the stratum corneum. Groups of 5 mice then transcutaneouslyvaccinated with CS3 (25 μg), CS6 (25 μg) with and without 10 μg ofLTR192G adjuvant. Patches were loaded with a 25 μl volume containing CS3or CS6 alone or CS3 plus LTR192G or CS6 plus LTR192G. The patches wereapplied overnight (˜18 hr). Separate groups of mice were injectedintradermal with 25 μg of CS3 or CS6. All mice received threevaccinations (day 0, 14 and 28). Sera were collected two weeks after thethird immunization and evaluated for antibodies to CS3, CS6 and LTR192G.

The results shown in FIG. 4 demonstrate that CS3 and CS6 elicit serumantibodies when epicutaneously applied to the skin. The serum antibodyresponse were further enhanced by co-administration of low doses ofLTR192G (10 μg). As depicted in FIG. 4, serum titers to CS3 and CS6 wereincreased 2-fold and 12-fold, respectively, by addition of LTR192Gadjuvant. Intradermal injection of CS3 elicited a high titer response(1:273,695) compared to epicutaneous application (1:30,581).Transcutaneous vaccination with CS6 elicited very high-titer antibodies(1:188,984), which were 10-fold greater compared to intradermalinjection of CS6 (1:17,036). In addition, high antibody titers toLTR192G were also detected whether the LTR192G was epicutaneouslyapplied alone or in combination with CS3 or CS6. These resultsdemonstrate that high molecular weight ETEC antigens (CS3, ˜3megadaltons and CS6 ˜1 megadalton) are immunogenic when administered ina patch on skin that has been pretreated to remove the stratum corneum.The magnitude of the immune response is greatly enhanced byco-administration of an adjuvant (LTR192). The mutant LTR192G alsoelicits production of high titer antibodies against itself whenepicutaneously applied alone or in combination with CS3 or CS6.

Example 2 Transcutaneous Vaccination with Divalent and TrivalentCombinations of ETEC Subunit Vaccines

Skin was pretreated as described in Example 1. Mice weretranscutaneously vaccinated with patches containing 25 μg CS3/10 μg LTRL192G; 25 μg CS6/10 μg LTR192G; or a cocktail of 25 μg CS3/25 μg CS6/10μg LTR192G. The results are shown in FIG. 5. These results demonstratethat the trivalent vaccine combination (CS3/CS6/LTR192G) elicited serumIgG titers that were comparable to the divalent vaccines (CS3/LTR192Gand CS6/LTR192G).

This clearly demonstrates the feasibility of combining multiple ETECsubunit vaccines into a single patch and that multiple subunits vaccinescan be co-administered without negatively affecting the magnitude of theimmune response to either subunit (i.e., CS3 or CS6)

Example 3 TCl with CS3/CS6 with and Without Co-Administered LTR192GAdjuvant

The next study was undertaken to determine if LTR192G would adjuvant theimmune response to an epicutaneously administered combination of CS3 andCS6. The animals were pretreated as described in Example 1 and thevaccine loaded patches were applied (overnight) to the pretreated skin(base of tail). The results shown in FIG. 6 demonstrate that the immuneresponses to CS3 and CS6 were greatly enhanced 5-fold and 24-fold,respectively, be addition of 10 μg LT to the trivalent mixture. Thisestablishes that it is feasible to transcutaneously vaccinate with threeETEC antigens. LTR192G is an important adjuvant and antigen, and itenhances the immune response to CS3 by 5-fold and CS6 by 24-fold.

Example 4 CS3 and CS6 Molecules are Antigenically Distinct

The specificity of the immune response to CS3 and CS6 was determined.Mice were pretreated with tape stripping and received two patches: oneon day 0 and the other on day 14. Groups of animals were vaccinated with25 μg CS3/10 μg LTR192G or with 25 μg CS6/10 μg LTR192G. Ten days afterthe second dose (day 24) serum was collected and evaluated forantibodies to CS3 and CS6. The results shown in FIG. 7 demonstrate thatvaccination with CS3 elicits specific antibodies that did not exhibitcross-reactivity with CS6. Likewise, antibodies to CS6 did not recognizeCS3. These results clearly show that immunity to CS3 and CS6 is highlyspecific and that ETEC vaccines intended for broad range protectionagainst enterotoxigenic E. coli strains will need to be multivalent.

Example 5 TCl with CS3 and LTR192G Elicits IgG1 and IgG2a SubclassAntibodies

Mice were pre-shaved at the base of their tails and the skin washydrated with 10% glycerol and 70% isopropyl alcohol. Their hydratedskin was pretreated with emery paper 10 times. Groups of mice werevaccinated with 25 μg CS3 alone or with a combination of 25 μg CS3/10 μgLTR192G. The mice were transcutaneously vaccinated on three times (day0, 14 and 28) and serum collected 30 days after the third immunization(day 58). The results shown in FIG. 8 demonstrate that IgG1 is the majorIgG subclass elicited here. IgG1 titers were greater when the LTR192Gadjuvant was co-administered with CS3. In addition, measurable antigenspecific IgG2a was also detected. IgG2a subclass, however, was onlydetected when the adjuvant was co-administered. These results confirmand extend the previous observation that the LTR192G adjuvant doesaugment the serum antibody response to CS3 and further demonstrates thatthe adjuvant may also direct Th2 and Th1 immune responses to antigensdelivered by the epicutaneous route.

Example 6 TCl with CS6 and LTR192G Elicits IgG1 and IgG2a SubclassAntibodies

The mice were shaved and pretreated as described in Example 5. Groups ofmice were transcutaneously vaccinated with 25 μg CS6 alone or with acombination of 25 μg CS6/10 μg LTR192G. The mice received threevaccinations (day 0, 14 and 28) and serum was collected 30 days afterthe third immunization. The results in FIG. 9 demonstrate that LTR192Gdid adjuvant (i.e., augment) the serum IgG response to CS6. As with CS3,antibodies to CS6 were both IgG1 and IgG2a subclasses. The generationsCS6-specific IgG2a, however, was dependent upon use of the LTR192Gadjuvant.

Example 7 Serum IgG Subclasses Elicited to LTR192G Following TCl

Mice were pretreated by the same procedure described in Examples 5 and6. The mice received three transcutaneousvaccinations (day 0, 14 and 28)and serum was collected 30 days after the third immunization (day 58).The results shown in FIG. 10 show that IgG1 was a major serum antibodysubclass elicited by TCl. As with CS3, CS6, LTR192G-specific IgG2asubclass was also elicited by TCl.

Example 8 Serum IgG Subclasses Elicited to LTR192G when Co-Administeredwith CS3 or CS6 in TCl

Mice were pretreated by the procedures described in Examples 5-7. Inthis study, 10 μg of LTR192G was admixed with 25 μg of CS3 or 25 μg CS6.Groups of mice received three transcutaneous vaccinations on day 0, 14and 28 and serum was collected 30 days after the third immunization (day58). As was observed in Example 7, serum IgG1 was the major subclassantibody elicited against LTR192G (FIG. 11). Measurable IgG2a was alsoproduced. This study further demonstrates that vaccination with acombination of CS3/LTR192G or CS6/LTR192G did not negatively affect theproduction of antibodies to the adjuvant. These results indicates thatas a component of the ETEC vaccine LTR192G serves a dual purpose, as anadjuvant and as an antigen in the vaccine.

The significance of IgG subclass characterization is related to themechanism by which a transcutaneous ETEC vaccine might protect againstnatural infection. These results demonstrate that transcutaneousvaccination elicits two subclasses of IgG antibody that are expected tofunction differently in protecting the host against natural infection.These mechanisms are by “neutralization” and “complement mediatedcytotoxicity.” IgG1 antibodies to CS3 and CS6, for example, are expectedto function by blocking (neutralizing) the ability of CS3⁺ and CS6⁺ ETECstrains from colonizing the small intesting, a step that is essential topathogenesis. IgG2a class antibodies are expected to protect the hostfrom infection by a different mechanism. This class of antibody, when incomplex with CS3 or CS6 antigens on the surface of enterotoxigenic E.coli, will mediate the activation of complement, which through a seriesof enzymatic steps, will result in the lysis (killing) of the bacterialcells. Both mechanism are effective in protecting the subject againstinfection.

Example 9 Mucosal Immune Response to CS3 Antigen After TCl

The mucosal (gastrointestinal) immune response elicited by TCl with ETECsubunit vaccines was characterized. A study was conducted to determineif TCl with CS3 with and without the LTR192G would result in theproduction of antibodies in gastric mucosa. Mice were shaved (48 hr inadvance) at the base of the tail, the skin hydrated and tape stripped 10times. Vaccine-loaded patches were placed over the pretreated skin.Groups of mice received patches with the following formulations:phosphate buffered saline (PBS); 25 μg CS3 alone; and 25 μg CS3/10 μgLTR192G. The patches were applied overnight. A separate group of micewas vaccinated by intradermal injection of 25 μg CS3. All mice receivedthree vaccinations on day 0, 14 and 28. Fresh fecal samples werecollected 7 days after the third immunization (day 35). Samples wereprocessed as described in Materials and Methods. Vaccination with CS3alone did not elicit antigen-specific antibody, with the exception ofone animal (FIGS. 12B and C). Mice vaccinated with CS3/LTR192G developeddetectable fecal IgG to CS3. Fecal IgA to CS3 was low level (FIGS. 12Cand G). Intradermal vaccination with CS3 resulted in measurable fecalIgA and IgG titers to CS3.

Example 10 Mucosal Immune Responses to CS6 Antigen After TCl

Mice were pretreated and immunized as described in Example 9. Groups ofmice were transcutaneously vaccinated with patches containing thefollowing formulations: phosphate buffered saline (PBS); 25 μg CS6; and25 μg CS6 with 10 μg LTR192G. The patches were applied overnight. Aseparate group of mice was immunized by intradermal injection with 25 μgCS6 alone. All mice received three vaccinations (days 14 and 28) andfecal samples were collected 7 days after the third immunization. Theseresults are shown in FIG. 13. Mice receiving CS6 alone developed littleor no detectable fecal IgA or IgG (panels B and F). Mice which weretranscutaneously vaccinated with CS6/LTR192G had low-titer, butmeasurable, CS6-specific IgA (panel C). The CS6-specific IgG was readilydetected in samples from all mice (panel G). Mice receiving intradermalCS6 did develop measurable antigen-specific IgA and IgG (panels D and H,respectively).

Example 11 Mucosal Immune Responses to LTR192G Antigen After TCl

Mice were pretreated and immunized as described in Example 9. Groups ofmice were transcutaneously vaccinated with patches containing thefollowing formulations: phosphate buffered saline (PBS); 10 μg LTR192Galone; 25 μg CS3/10 μg LTR192G; and 25 μg CS6/10 μg LTR192G. The patcheswere applied overnight. A separate group of mice was immunized byintradermal injection with 25 μg CS6 alone. All mice received threevaccinations (day 0, 14 and 28) and fecal samples were collected 7 daysafter the third immunization. These results are shown in FIG. 14. Micereceiving LTR192G alone by TCl developed measurable fecal IgA and IgG(panels B and F). Mice transcutaneously vaccinated with CS3/LTR192Gdeveloped measurable LTR192G-specific IgA (panel C) and fecal IgG (panelG). Mice which were transcutaneously vaccinated with CS6/LTR192Gdeveloped measurable fecal IgA and IgG to LTR192G (panels D and H).These results support the dual role of the LTR192G in a multivalent ETECvaccine as a potent adjuvant for boosting systemic and mucosal immuneresponses to colonization factor antigens and as an vaccine forheat-labile enterotoxin, a toxin responsible for clinical disease.

Example 12 TCl with Divalent ETEC Vaccines Elicits Antigen-SpecificAntibody Secreting Cells (ASC) in the Spleen

Antigen-specific B cells were next detected in the spleen of vaccinatedmice. Mice were pretreated and transcutaneously vaccinated as describedin Example 9. Groups of mice were vaccinated with one of the followingformulations: 25 μg CS3; 25 μg CS3/10 μg LTR192G; 25 μg CS6; or 25 μgCS6/10 μg LTR192G. The patches were applied overnight to the pretreatedskin. Separate groups of mice were vaccinated by intradermal injectionat the base of the tail with 25 μg CS3 and 25 μg CS6. All mice receivedthree vaccinations (day 0, 14 and 28). Spleens were collected 30 daysafter the third immunization (day 58) and single-cell suspensions wereprepared and the cells cultured and stained for identification of Bcells producing antigen-specific IgG (IgG-ASC) and IgA (IgA-ASC) asdescribed in the Materials and Methods. No IgA- or IgG-ASCs weredetected in spleens from mice transcutaneously immunized with CS3 alone(FIG. 15, panels A and B) or CS6 alone (FIG. 15, panels C and D). Incontrast, mice transcutaneously immunized with CS3/LTR192G orCS6/LTR192G did develop CS3- and CS6-specific IgA- and IgG-ASCs,indicating that the generation of ASC's in the spleen was dependent uponco-administration of the adjuvant. In addition, LTR192G specific IgA-and IgG-ASC were present in spleen cell suspensions of mice vaccinatedwith the divalent vaccines (i.e., CS3/LTR192G and CS6/LTR192G). Theseresults demonstrate that the generation of antigen-specific B-cellimmunity by TCl is dependent upon co-administration of LTR192G for theseantigens.

Example 13 TCl with Trivalent ETEC Vaccine (CS3, CS6 and LTR192G)Elicits Antigen-Specific Antibody Secreting Cells in the Spleen

The purpose of this study was to characterize the B-cell response in thespleen after TCl with a trivalent ETEC vaccine. Mice were pretreated andvaccinated as described in Example 12. Mice were vaccinated with patchescontaining a cocktail of 25 μg CS3/25 μg CS6/10 μg LTR192G. The patcheswere applied overnight and all mice received three transcutaneousvaccinations (day 0, 14 and 28). Spleens were collected 30 days afterthe third immunization (day 58). The splenocytes were cultured andantigen-specific ASC stained and counted as described in the Materialsand Methods. The results are shown in FIG. 16. Mice vaccinated with thetrivalent vaccine (CS3, CS6 and LTR192G) were found to have generatedIgA-ASC (panel A) and IgG-ASC (panel B) for each of the ETEC antigens inthe vaccine. These results demonstrate that it is feasible totranscutaneously vaccinate with a mixture of ETEC subunit antigens andto elicit clonal expansion of antigen-specific B cells within thespleen.

Example 14 TCl with Monovalent and Divalent ETEC Subunit VaccinesElicits Antigen Specific Antibody Secreting Cells in Lymph Nodes

It was hypothesized that transcutaneous vaccination at the base of thetail may result in clonal expansion of B cells within lymph nodes thatdrain the dorsal caudal skin surface. To test this hypothesis, mice wereepicutaneously immunized at the base of the tail on skin that had beentape stripped 10 times. Groups of mice received three doses of thefollowing formulations: 25 μg CS3; 25 μg CS6; 25 μg CS3/10 μg LTR192G;and 25 μg CS6/10 μg LTR192G. The patches were applied overnight.Separate groups received intradermal injections of 25 μg CS3 or 25 μgCS6. All groups were vaccinated three times (day 0, 12 and 28) and theinginual lymph nodes collected 30 days after the third immunization.Single cell suspensions were prepared and cultured with antigens thatwere plated onto the microwells. IgG- and IgA-ASC were visualized bystaining as described in Materials and Methods. The results shown inFIG. 17 demonstrate that generation of CS3- or CS6-specific IgG-ASC(panels A and B, respectively) was dependent upon co-administration ofthe adjuvant. It was also observed that CS3, CS6 and LTR192G specificASC's were more numerous in the inguinal lymph node than in spleen (FIG.15). This is consistent with the hypothesis that skin immunizationlikely involves the mobilization and activation of resident skinLangerhans cells to become activated by LTR192G interaction with the GM1receptor. Antigen loaded and activated Langerhans cells are believed toegress from the epidermis, migrate through the dermis, and into thedrain lymphatics. The antigen laden Langerhans cell takes up residencein the lymph node were B cell clonal expansion takes place, hence therelative abundance of CS3, CS6 and LTR192G within the draining inguinallymph node.

Example 15 Generation of B-Cell Immunity to Complex Mixtures of ETECAntigens Delivered by TCl

We also demonstrate that TCl is suitable for immunizing with a complexmixture of antigens. Mice were pretreated and immunized as described inExample 14. The patch was loaded with a cocktail of three ETEC antigensin the following formulation: 25 μg CS3, 25 μg CS6 and 10 μg LTR192G.After three epicutaneous immunizations, the inguinal lymph nodes werecollected and CS3, CS6 and LTR192G specific IgG-ASC was determined. FIG.18 shows TCl with the trivalent ETEC vaccine did stimulate thegeneration of ASC specific for each of the subunit antigens in thevaccine.

Example 16 Transcutaneous Vaccination with CFA/I Elicits SystemicImmunity

Having established that it is possible to immunize via the skin withmixtures of high molecular weight antigens, we next investigated thefeasibility of TCl with a tetravalent ETEC vaccine. CFA/I is widelyexpressed by ETEC strains isolated throughout the world. It is estimatedthat 30% of all human ETEC strains express CFA/I. Initially, wedetermined if CFA/I was immunogenic when delivered into the skin. Inthese studies, mice were shaved at the base of the tail, the skinhydrated and gently abraded with emery paper 5 times to disrupt thestratum corneum. The patch was loaded with one of the followingformulations: 25 μg CFA/I or 25 μg CFA/I/10 μg LTR192G. The patches wereapplied overnight. A separate group was immunized by intradermalinjection with 25 μg CFA/I. All mice received two doses (day 0 and 14)and serum was collected for analysis 10 days after the secondimmunization (day 24). As shown in FIG. 19, mice transcutaneouslyvaccinated with CFA/I did develop serum antibodies after twoimmunizations (panel A). Co-administration with LTR192G increased theserologic response approximately 8-fold, indicating that LTR192G is ageneral adjuvant for stimulating immune responses to antigens presentedin the skin. As expected, transcutaneously vaccinated mice alsodeveloped antibodies to LTR192G (panel B).

Example 17 Transcutaneous Vaccination with CFA/I Elicits MucosalImmunity

Fresh fecal samples were collected from mice that had beentranscutaneously immunized with CFA/I in Example 16. The fecal sampleswere tested for CFA/I-specific IgA and IgG. As shown in FIG. 20, CFA/Ialone did not elicit the generation of detectable fecal IgA (panel B) orfecal IgG (panel F). Mice that received CFA/I and LTR192G did producefecal IgA (panel C) and IgG (panel G) following vaccination.Interestingly, mice that received CFA/I by intradermal injection did notgenerate fecal antibodies to the antigen (panels D and H).

Examples 16 and 17 demonstrate that it is feasible to transcutaneouslyvaccinate with CFA/I and that production of a systemic and mucosalresponses that were, to great extent, dependent upon co-administrationof the adjuvant.

Example 18 Transcutaneous Vaccination with a Tetravalent ETEC VaccineElicits Systemic Immunity

Broad coverage by a vaccine to prevent ETEC infection will require theuse of a multivalent vaccine. Epidemiology studies demonstrate that abroad coverage (80%-90% of ETEC strains) ETEC vaccine will likelyrequire a combination of at least three colonization factor antigenswith E. coli heat-labile enterotoxin (LT). The following studies wereconducted to demonstrate the feasibility of transcutaneous vaccinationwith a tetravalent vaccine. Mice were preshaved about 48 hr before TCland the skin hydrated with 10% glycerol and 70% isopropyl alcohol. Thevaccination site was pretreated with emery paper 5 times and the vaccineloaded patch applied to the skin. The formulation used here was 25 μgCFA/I, 25 μg CS3, 25 μg CS6 and 10 μg LTR192G. The mice were vaccinatedtwice on day 0 and 14 and serum was collected for evaluation 10 daysafter the second immunization (day 24). The results in FIG. 21 show thatall four components of the vaccine elicited serum responses after twoimmunizations. It should also be pointed out that the LTR192G-adjuvantdose (10 μg) did not need to be increased further in order to achieveimmunization. This observation is significant since it clearly indicatesthat complex mixtures of subunit vaccines can be delivered via the skinand that the adjuvanting activity of the LTR192G does not requirefurther increase in the dose of the adjuvant. Furthermore, theantigenicity of LTR192G was not diminished by co-administration withmultiple antigens (CS3, CS6 or CFA/I).

Example 19 Transcutaneous Vaccination with a Tetravalent ETEC VaccineElicits Mucosal Immunity to Colonization Factor Antigens

Mice were pretreated as described in Example 18. Groups of mice werevaccinated with patches containing the following formulation: 25 μgCFA/I, 25 μg CS3, 25 μg CS6 and 10 μg LTR192G. All mice weretranscutaneously vaccinated at the base of the tail on day 0, 14 and 28.Fecal samples were collected two weeks after the third immunization (day42). The samples were processed and evaluated for antibodies to each ofthe antigens in the vaccine. The results shown in FIG. 22 demonstratethat fecal IgA and IgG were elicited to CFA/I (panels A and D), CS3(panels B and E), and CS6 (panels C and F).

Example 20 TCl with a Tetravalent ETEC Vaccine Elicits Anti-Toxin (LT)Immunity

Fecal samples were collected from mice that were vaccinated as inExamples 18 and 19. The samples were processed as described in Materialsand Methods and evaluated for fecal IgA and IgG to LTR192G. As shown inFIG. 23, LTR192G elicited significant titers of antigen-specific IgA andIgG antibodies, whether the adjuvant was administered alone (panels Aand D), with CFA/I (panels B and E), or as a tetravalent cocktail(panels C and F).

These above examples clearly demonstrate that multivalent vaccines canbe efficiently delivered via the skin without the use of a hypodermicneedle, jet injector, or other barrier disruptors. These examples alsodemonstrate the LT adjuvant has a significant role in stimulating theproduction of high titer serum antibodies; directing the humoralresponse by mediating the production antigen-specific IgG2a and IgG1;and it is essential in some cases to promoting a mucosal antibodyresponse to ETEC colonization factor antigens. We have demonstrated thatmultivalent vaccine are effectively delivered by TCl without deleteriouscompetition among the cocktail of antigens. This is important in thetreatment of diseases caused by pathogens which express a variety ofantigenic specificities because assuring coverage of the many differentisolates will probably require including four, five, six, seven, eightor more antigens in a vaccine. Neutralization of toxin and prevention ofinfection provides treatment (therapeutic and/or prophylactic) at twocritical points of host-pathogen interaction. This is an unexpectedimprovement over the prior art.

Example 21 TCl Elicits Long-Lived Neutralizing Antibodies Against LT

Human volunteers were enrolled into a phase I clinical trial. Thevolunteers were transcutaneously immunized on the skin over the deltoidmuscle. The skin was pretreated with isopropyl alcohol and hydrated witha mixture of 10% glycerol and 70% isopropyl alcohol. A gauze patch (4×4inches) was affixed to an adhesive backing and an aqueous solution of LTwas applied to the patch. The wet patch formulations were in PBS with 5%lactose containing one of the following amounts of LT: 50 μg, 100 μg,250 μg or 500 μg. The volunteers received two doses (day 0 and 30).Serum was collected prior to immunization (pre-immune) and 312 daysafter the first immunization (post-immunization). By ELISA, the seraexhibited antibody titers to LTR192G that were above their pre-immunetiters (Table 6).

TABLE 6 Detection of serum antibodies after two rounds of TCI Endpointserum Fold increase antibody titer to LT neutralizing titer inneutralizing Volunteer LT (ELISA units¹) in CHO cell culture² antibody(day number Day 0 Day 312 Day 0 Day 312 312/day 0) 12 583 10271 <1:21:16 8 13 1079 5098 <1:2 1:16 8 15 804 13468 <1:2 1:16 8 16 1231 12317<1:2 1:8 4 18 671 18301 <1:2 1:16 8 19 730 8238 <1:2 1:8 4 ¹ELISA unitis the endpoint titer that is equal to 1 OD at 405 nm ²Lowest serumdilution that blocked (neutralized) CHO cell elongation by >90%

An in vitro CHO cell assay was used to determine if these sera containedantibodies that would neutralize LT receptor binding and in vitrotoxicity. All sera were found to have antibodies that blocked(neutralized) LT toxicity in vitro. This result shows that TCl doeselicit antibodies that function to neutralize the toxic effects ofheat-labile enterotoxin LT on cells. TCl elicits long-lived immunity toLT and the antibodies induced thereby neutralize the toxin.

Example 22 Transcutaneous Vaccination with CS3 and LTR192G SubunitVaccines Elicit Serum Antibodies that Recognize Antigens on CS3⁺ETECWhole Cells

We next show that antibodies elicited by transcutaneous vaccination areimmunoreactive with native conformational epitopes expressed byenterotoxigenic E. coli organism. Mice were pre-shaved and the skinhydrated. The patch was prepared by directly applying an aqueoussolution (25 μl) consisting of 25 μg CS3 and 10 μg LTR192G to the gauzepatch. While the skin was still hydrated, the patch was applied andmaintained in place for ˜18 hr. A group of 10 mice received two patcheson day 0 and 14. Serum was collected 10 days after the secondimmunization on day 24. These sera were evaluated by the ELISA methodfor antibodies to purified CS3, LTR192G and CS3 expressingenterotoxigenic E. coli (strain E243778). The results shown in FIG. 24demonstrate that the immunized mice developed significant antibodytiters to purified CS3 (1:70,464) and LTR192G (1:32,657). These serawere also found to have significant serum antibodies titers to ETECwhole cells (1:11,206). These results demonstrate that TCl with ETECsubunit vaccines (e.g., CS3 and LTR192G) does result in the productionof antibodies that recognize native conformational determinants on aCS3-expressing ETEC strain.

Example 23 TCl with Killed ETEC Elicits Immunity Against the BacterialPathogen

It is feasible to transcutaneously vaccinate with a killed bacterialorganism applied to the skin. ETEC (strain E243778) was grown on abacterial growth media. The bacteria were harvested from the cultures bycentrifugation and washed with phosphate buffered saline. A 2% solutionof formalin was added to cell pellet and cells suspended to a singlecell suspension. The cells were mixed overnight at room temperature inorder kill all bacteria. The killed cells were washed and resuspended inPBS. Mice were shaved and the skin at the base of the tail pretreated bytape stripping 10 times to remove the stratum corneum. The gauze patchwas loaded with 25 μl volume containing 10⁹ killed ETEC whole cells(EWC) and 10 μg LTR192G was added to the mixture. The patch was appliedovernight. A group of 10 animals were vaccinated on day 0 and 14 and theserum collected 10 days after the second immunization. The serum wasevaluated for antibodies to EWC and LTR192G using the ELISA methoddescribed in Materials and Methods. The results are depicted in FIG. 25.All mice were found to have serologically converted and to have serumantibodies to the bacterial whole cells (1:324) as well as LTR192G(1:21,044). These results are the first demonstration that it isfeasible to transcutaneously vaccinate with killed bacterial whole cellsand to elicit an organism-specific immune response.

Example 24 TCl with ETEC Colonization Factor, Heat-Labile Enterotoxin(LT), and Heat-Stable Toxin (ST) Antigens

Certain strains of enterotoxigenic E. coli are known to produce a secondenterotoxin called heat-stable enterotoxin (ST). Like the heat-labileenterotoxin (LT), ST is highly reactogenic in humans and it is a causeof severe diarrhea in children and adults. Strains of ETEC may produceSTa alone, LT alone, or a combination of STa and LT. STa is a smallmolecular weight peptide 19 amino acids and contains 6 cysteines. STa isknown to poorly immunogenic when administered by injection. This studywas conducted to demonstrate that it is possible to transcutaneouslydeliver a complex ETEC vaccine that consists of multiple colonizationfactors (CS3 and CS6) and both enterotoxins (LT and ST). Mice werepretreated by tape stripping 10 times at the base of the tail using themethod described herein. The patch formulations used here were thefollowing: 25 μg CS3/25 μg CS6; 25 μg CS3/25 μg CS6/10 μg LTR192G; and25 μg CS3/25 μg CS6/10 μg LTR192G/8 μg ST. Mice received threetranscutaneously vaccinations on day 0, 14 and 28 and serum collectedtwo weeks after the third immunization. The results shown in FIG. 26demonstrate that immunity to the colonization factors was significantlyenhanced by addition of LTR192G. This study also demonstrates that theaddition of ST can be added to the multivalent ETEC vaccine withoutadversely affecting the adjuvanting action of LTR192G or the generationof the immune response to these colonization factors. It suggests thatit will be possible to develop a pentavalent ETEC vaccine consisting ofCS3, CS6, CFA/I, LT and STa or any other combination of antigens. Theseantigens can be safely administered via TCl without toxicity or serious,adverse side affects.

Example 25 Wet, Protein-In-Adhesive and Air-Dried Patch Formulations forDelivery of ETEC Antigens

Patches are versatile vehicles for delivering ETEC vaccines by TCl.Here, LT was formulated in four different ways. First, LT (10 μg) wasformulated in an aqueous solution consisting of neutral pH phosphatebuffered saline containing 5% (w/v) lactose. This formulation wasapplied directly to skin hydrated with 10% glycerol and 70% isopropylalcohol. The solution was left undisturbed or was over laid with a gauzepad for 1 hr. Second, LT was blended with various adhesives (e.g.,Klucel). The formulation was then spread as a thin coat over anocclusive backing. The LT was spread with a Rotograveur press as a finefilm to an effective concentration of 10 μg in a 1 cm² area. The filmwas air-dried at room temperature and moisture content ranged betweenless than 0.2% to 5% water. Patches (˜1 cm²) were punched from thesheet. The patches were stored at ambient temperature and 4° C.exhibited the same delivery characteristics. Third, LT was directlyapplied to a gauze pad and spread evenly over the surface to aconcentration of 10 μg/cm². These patches were air-dried overnight.Fourth, LT (10 μg in 25 μl PBS and 5% lactose) in an aqueous formulationwas dropped directly onto a gauze pad (˜1 cm²) that was affixed to anadhesive backing. These patched were air-dried overnight at ambienttemperature. These patches were stored at 4° C. for one month prior touse.

The patches were compared for delivery of LT antigen using the mousemodel described in Materials and Methods. The shaved skin at the base ofthe tail was hydrated and pretreated with a pumice-containing swab(formulated with 10% glycerol and 70% isopropyl alcohol) to disrupt thestratum corneum. Groups of 5 mice received two patches: one on day 0 andthe other on day 14. The air-dried patch was re-hydrated with 25 μl ofwater prior to application. The patches were removed after 24 hr. Forthe liquid formulation, the LT containing solution was left on the skinfor 1 hr prior to rinsing with water to remove excess LT. Serum wascollected from each animal 2 weeks after the second immunization (day28). The results are shown in FIG. 27. These results demonstrate thatall methods were suitable for transcutaneous delivery of LT across theskin. This example shows that the patch formulation may be an aqueousliquid that is applied directly to skin and over laid with a patch; adry patch with the antigens incorporated within the adhesive(protein-in-adhesive) and spread as a thin coating over an occlusivebacking; a patch in which the antigens are applied in solution(separately or as a cocktail) directly to a suitable surface and allowedto air-dry; or as a hydrated patch in which the antigens are in asolution and the appropriate amount of the solution is directly appliedto patch surface shortly before applying the patch.

Example 26 Protein-In-Adhesive Formulations for Transcutaneous Deliveryof ETEC Subunit Vaccines and Enterotoxins

The protein-in-adhesive formulations are intended to incorporate one ormore ETEC subunit antigens into an adhesive formula. The formula is alsosuitable for incorporating killed ETEC whole cells (˜10⁴ to 10⁸ killedbacteria per dose) with or without LT-adjuvant. The blend is then castover a sheet of occlusive (or semi-occlusive) backing as a thin film.The vaccine/adhesive mixture is allowed to cure (room temperature or 40°C.) until the film is dry (water content may vary between 0.5% and 5%;1-2% is desired). The cast film is cut from the die-cast to the desiredsize and shape. The dry patches are then sealed in a light-tight,waterproof plastic or foil pouch. Patches produced in this manner may bestored refrigerated or at ambient temperatures. The protein-in-adhesiveis flexible in that the multivalent vaccine blend may be varied toincorporate different amounts and ratios of one or multiple antigens andadjuvant. In addition, the patch size may be varied in order to adjustdosing. Depending upon the age of the individual, patch size (dose) canbe varied for use in children and adults.

Protein-in-adhesive formulations are flexible and uniquely allow thevaccines to be coated in layers. These patches are manufactured in amanner wherein each vaccine component is layered separately onto thepatch backing. The objective is to create a multilaminar membrane werecomponent 1 is layered onto the backing, component 2 film is layeredover 1, component 3 is layered on top of components 1 and 2, andcomponent 4 is the outermost layer. The advantage of this approach isthat it provides flexibility to the formulation (i.e., patches may beproduced from the same process using different ratios of antigen andadjuvant or in the case were the vaccine is manufactured to contain onlyone or two components). This multilayered formula also has the advantageof controlling the release rates of each antigen and the adjuvant. Insome instances, it will be desirable to have the LT-adjuvant releasedimmediately in order to pre-prime the skin dentritic cells (Langerhanscells) prior to release of other antigens. In such formulations, theLT-primed Langerhans cells may more efficiently capture and process thetoxin and colonization factor antigens. Controlled delivery is a moreefficient use of the adjuvant and antigens and will allow the doses tobe further reduced.

Tables 7-8 describe formulations that may be suitable for stabilizingadjuvants and/or antigens into an adhesive. The following are intendedto be examples of such formulations and are not intend to restrict theformulation.

TABLE 7 Eudragit EPO formulation Dry Wt Ingredients % NVC Wt (gm) Wt (%)(gm) % (w/w) EPO polymer 100 22.8 22.0 22.8 58.8 Succinic acid 100 1.01.0 1.0 2.6 ATBC 100 15.0 14.5 15.0 38.7 Water 0 55.0 62.6 0 0 Totals104 100 38.8 100

TABLE 8 Adhesive formulation with E. coli CS6 and LT Wet Weight g g (natDry Wt Dry Ingredients % NVC (nat/water) only) Wet % (gm) (% w/w) 1 ×PBS 6.0 15.6 15.6 38.2 0.9 13.3 EPO 37.5 15.6 15.6 38.2 5.9 82.3Natrasol 2.5 9.0 0.2 22.4 0.2 3.2 CS6 100 0.05 0.05 0.12 0.05 0.66 LT100 0.02 0.01 0.04 0.02 0.2 Tween 20 100 0.03 0.03 0.07 0.03 0.4

Example 27 Gel Formulations for Delivery of ETEC Subunit Vaccines (CS3,CS6, CFA/I, ST and LT) and Killed ETEC Whole Cells

Gels are examples of fully hydrated or wet patches. These formulationsare intended to incorporate one or more ETEC subunit antigens entrappedwithin a gel matrix. This formulation is also suitable fortranscutaneous delivery of killed ETEC whole cells (˜10⁴ to 10⁸ killedbacteria per dose) with or without LT. The vaccines are formulated byblending a solution containing the antigens in the desired amounts andratios with a carbomer, pluronic, or a mixture of the two gel components(see below). The gel containing vaccine is then coated onto a strip ofthe material that holds the gel in-place without spilling. It isimportant that the material have a low binding capacity for the proteinsin the vaccine. The strip may comprise materials such as polymers,natural and synthetic wovens, non-wovens, foil, paper, rubber, orcombinations thereof. The strip may be a single layer or a laminate ofmore than one layer. Generally, the strip is substantially waterimpermeable and helps to maintain the skin in hydrated condition. Thematerial may be any type of polymer that meets the required flexibilityand low binding capacity for proteins. Suitable polymers include, butare not limited to, polyethylene, ethyl vinylacetate, ethylvinylalcohol, polyesters, or Teflon. The strip of material for holding thegel is less than 1 mm thick, preferably less than 0.05 mm thick, mostpreferably 0.001 to 0.03 mm thick.

The gel-loaded strip may be of different sizes and shapes. It ispreferred that the corners be rounded for ease of application-. Thelength of the strip can vary and is dependent upon the intended user(i.e., children or adults). It may be from about 2 cm to about 12 cm,and is preferably from about 4 cm to about 9 cm. The width of the stripwill vary but it may be from about 0.5 cm to about 4 cm.

The strip may contain shallow pockets or dimples. To hold in place, whenthe vaccine containing gel is coated onto the strip, the gel should fillthe shallow pockets that provide reservoirs for the gel. The shallowpockets may be about 0.4 mm across and about 0.1 mm deep. The gel-loadedpatch is about 1 mm thick, with a preferred thickness of about 0.5 mm orless.

The flexural stiffness is important since maximal contact between thegel and the skin must be maintained. The strip will need to conform tothe contour of the anatomical location where the patch is applied (e.g.,skin over the deltoid muscle, volar forearm, neck, behind the ear, orother locations). Flexural stiffness can be measured with aHandle-O-Meter (Thwing Albert Instruments). The flexural stiffnessshould be less than 5 gm/cm, more preferably less than 3 gm/cm. Therelatively low stiffness enables the strip of material to drape over thecontoured surface with little force being exerted.

The gel-loaded strip is held in place by affixing the strip to anadhesive backing with the gel surface facing away from the adhesivebacking. The backing material may be occlusive or semi-occlusive (e.g.,TEGADERM). The backing is designed to hold the patch in place, to aid inmaintaining maximal contact between the skin and gel, and to prevent thegel from dehydrating during wear.

To prevent dehydration of the wet patch during storage and handling, itmay be placed on an inert plastic strip, which is fairly rigid. The gelsurface would be in direct contact with the plastic strip, and thegel/plastic interface has low peel force making it easy to separate thegel strip from the plastic strip. The plastic strip may be made ofpolyethylene or similar material. The gel-patch can be packaged in alight-proof and water tight plastic or foil pouch. The pouch can bestored refrigerated or at room temperature.

The following are intended as examples of the hydrated gel formulationand are not intended to restrict it: gels in phosphate buffered saline;1% CARBOMER 1342; 1.5% CARBOMER 940; 1.5% CARBOMER 934; 1.5% CARBOMER940, 2% sucrose, 10% isopropyl alcohol, 10% glycerol; 50% PLURONIC F87;and 30% PLURONIC F108.

Carbomer polymers are high molecular weight, acrylic acid-based polymersthat may be cross-linked with allyl sucrose or allylpentaerythritol,and/or modified with C10-C30 alkyl acrylates. These may or may or not beincorporated into a patch or may be delivered by other means know in theart into the skin.

Formulations may be comprised of carbomers of different averagemolecular weights. For example, the polymers may be CARBOMER 1342 (e.g.,1% CARBOMER 1342, 0.6 mg/ml LT, 0.3% methylparaben, 0.1% propylparaben,2.5% lactose, 1× PBS); CARBOMER 940 (e.g., 1.5% CARBOMER 940, 0.6 mg/mlLT, 0.3% methylparaben, 0.1% propylparaben, 2.5% lactose, 1× PBS). Eachformulation can be prepared in a phosphate buffered saline solution andcontain LT at a concentration of about 0.6 mg/ml or less, but antigensand adjuvants may also be formulated from about 0.001 mg/ml to about 0.6mg/ml or from about 0.6 mg/ml to about 6 mg/ml. In addition,antimicrobial agents such as methylparaben and propylparaben may beincluded.

Combinations of CARBOMER 940 and PLURONIC F87 (e.g., 1.5% CARBOMER 940,0.5% PLURONIC F87, 0.6 mg/ml LT, 0.3% methylparaben, 0.1% propylparaben,2.5% lactose, 1× PBS) may be used. PLURONICS are another class ofhydrogel that contain repeating segments of ethylene oxide-propyleneoxide-ethylene oxide. The amount of LT and antimicrobial agents in theformulation may be identical.

Other formulations may enhance delivery using penetration enhancers andcarbomers. For example, a gel may comprise CARBOMER 940 with PHARMASOLVE(e.g., 1.5% CARBOMER 940, 10% PHARMASOLVE, 0.6 mg/ml LT, 0.3%methylparaben, 0.1% propylparaben, 2.5% lactose, 1× PBS) while the finalgel may contain CARBOMER 940, glycerol, and isopropanol (e.g., 1.5%CARBOMER 940, 10% glycerol, 10% isopropanol, 0.6 mg/ml LT, 0.3%methylparaben, 0.1% propylparaben, 2.5% lactose, 1× PBS). Theconcentration of LT and antimicrobial agents may remain identical to theprevious formulations, or may be in other ranges specified.

Example 28 Dosages for CS3, CS6, CFA/I, ST and LT

The dose range may vary and may be dependent upon the age and medicalcondition of the subject. Doses of 1 mg to less than 5 μg may elicitantigen-specific immune responses in both human and animal subjects. Thedesired adult dose may range for colonization factors from about 1 μg toabout 100 μg of each (e.g., CS3, CS6 and CFA/I); the preferred dose ofeach colonization factor is from about 5 μg to about 50 μg. The desiredadult dose may range for LT from about 1 μg to about 100 μg; thepreferred dose of LT is from about 5 μg to about 50 μg. The desiredadult dose may range for ST (not conjugated to a carrier protein) fromabout 1 μg to about 100 μg. Since ST is poorly immunogenic, the adultdose may be from about 25 μg to about 100 μg. If ST is chemicallycoupled to LT (LT-ST), the ST equivalent dose may be from about 5 μg toabout 50 μg. Immunogenicity can be improved by conjugating ST to othercarrier proteins, including, for example, albumins, KLH or aggregatedantibodies. In this latter case, the dose of ST may be from about 5 μgto about 50 μg.

REFERENCES

-   Ahren et al. (1998) Intestinal immune responses to an inactivated    oral enterotoxigenic Escherichia coli vaccine and associated    immunoglobulin A responses in blood, Infect Immun., 66:3311-3316.-   Beebe et al. (1972) Long-term mortality follow-up of Army recruits    who received adjuvant influenza virus vaccine in 1951-1953, Am J    Epidemiol, 95:337-346.-   Beignon et al. (2001) Immunization onto bare skin with heat-labile    enterotoxin of Escherichia coli enhances immune responses to    coadministered protein and peptide antigens and protects mice    against lethal toxin challenge, Immunol, 102:344-351.-   Berardesca & Maibach (1988) Contact dermatitis in blacks, Dermatol    Clin, 6:363-368.-   Black (1993) Epidemiology of diarrhoeal disease: Implications for    control by vaccines, Vaccine, 11:100-106.-   Caeiro et al. (1999) Improved detection of enterotoxigenic    Escherichia coli among patients with travelers' diarrhea, by use of    the polymerase chain reaction technique, J Infect Dis,    180:2053-2055.-   Cassels & Wolf (1995) Colonization factors of diarrheagenic E. coli    and their intestinal receptors, J Industr Microbiol, 15:214-226.-   Cassels et al. (1992) Analysis of Escherichia coli colonization    factor antigen I linear B-cell epitopes, as determined by primate    responses, following protein sequence verification, Infect Immun,    60:2174-2181.-   Cheney et al. (1980) Species specificity of in vitro Escherichia    coli adherence to host intestinal cell membranes and its correlation    with in vivo colonization and infectivity, Infect Immun,    28:1019-1027.-   Clemens et al. (1988) Cross-protection by B subunit-whole cell    cholera vaccine against diarrhea associated with heat-labile    toxin-producing enterotoxigenic Escherichia coli: results of a    large-scale field trial, J Infect Dis, 158:372-377.-   Clements et al. (1980) Properties of homogeneous heat-labile    enterotoxin from Escherichia coli, Infect Immun, 29:91-97.-   Clements et al. (1988) Adjuvant activity of Escherichia coli    heat-labile enterotoxin and effect on the induction of oral    tolerance in mice to unrelated protein antigens, Vaccine, 6:269-277.-   Cohen et al. (2000) Safety and immunogenicity of two different lots    of the oral, killed enterotoxigenic Escherichia coli-cholera toxin B    subunit vaccine in Israeli young adults, Infect Immune 68:4492-4497.-   Craig (1966) Preparation of the vascular permeability factor of    Vibrio cholerae, J Bacteriol, 92:793-795.-   Cravioto et al. (1990) Risk of diarrhea during the first year of    life associated with initial and subsequent colonization by specific    enteropathogens, Am J Epidemiol, 131:886-904.-   Crottet et al. (1999) Expression, purification and biochemical    characterization of recombinant murine secretory component: a novel    tool in mucosal immunology, Biochem J, 341:299-306.-   Cryz & Gluck (1998) Immunopotentiating reconstituted influenza    virosomes as a novel antigen delivery system, Dev Biol Stand,    92:219-223.-   Czerkinsky et al. (1988) A novel two colour ELISPOT assay. I.    Simultaneous detection of distinct types of antibody-secreting    cells, J Immunol Meth, 115:31-37.-   Daniels et al. (2000) Traveler's diarrhea at sea: Three outbreaks of    waterborne enterotoxigenic Escherichia coli on cruise ships, J    Infect Dis, 181:1491-1495.-   Division of Health Promotion and Disease Prevention and Division of    International Health, I.O.M. (1986) The prospects for immunizing    against Escherichia coli (ETEC), pp.178-185, New Vaccine    Development: Establishing Priorities, vol. 2, National Academy    Press, Washington, D.C.-   Edelman (1980) Vaccine adjuvants, Rev Infect Dis, 2:370-383.-   El-Ghorr et al. (2000) Transcutaneous immunisation with herpes    simplex virus stimulates immunity in mice, FEMS Immunol Med    Microbiol, 29:255-261.-   Evans et al. (1977) Hemagglutination of human group A erythrocytes    by enterotoxigenic Escherichia coli isolated from adults with    diarrhea: correlation with colonization factor, Infect Immun,    18:330-337.-   Evans et al. (1988) Non-replicating oral whole cell vaccine    protective against enterotoxigenic Escherichia coli (ETEC) diarrhea:    stimulation of anti-CFA (CFA/I) and anti-enterotoxin (anti-LT)    intestinal IgA and protection against challenge with ETEC belonging    to heterologous serotypes, FEMS Microbiol Immunol, 1:117-125.-   Forrester & Ury (1969) The Signed-Rank (Wilcoxon) test in the rapid    analysis of biological data, Lancet, 1:239-241.-   Freedman et al. (1998) Milk immunoglobulin with specific activity    against purified colonization factor antigens can protect against    oral challenge with enterotoxigenic Escherichia coli, J Infect Dis,    177:662-667.-   Freytag & Clements (1999) Bacterial toxins as mucosal adjuvants,    Curr Topics Microbiol Immunol, 236:215-236.-   Fujita & Finkelstein (1972) Antitoxic immunity in experimental    cholera: a comparison of immunity induced perorally and parenterally    in mice, J Infect Dis, 125:647-655.-   Gilligan (1999) Escherichia coli. EAEC, EHEC, EIEC, ETEC, Clin Lab    Med, 19:505-521.-   Glenn et al. (1998a) Skin immunization made possible by cholera    toxin, Nature, 391:851.-   Glenn et al. (1998b) Transcutaneous immunization with cholera toxin    protects mice against lethal mucosal toxin challenge, J Immunol,    161:3211-3214.-   Glenn et al. (1999) Transcutaneous immunization with bacterial    ADP-ribosylating exotoxins as antigens and adjuvants, Infect Immun,    67:1100-1106.-   Glenn et al. (2000) Transcutaneous immunization: A human vaccine    delivery strategy using a patch, Nat Med, 6:1403-1406.-   Gockel et al. (2000) Transcutaneous immunization induces mucosal and    systemic immunity: A potent method for targeting immunity to the    female reproductive tract, Mol Immunol, 37:537-544.-   Hall et al. (2001) Induction of systemic antifimbria and antitoxin    antibody responses in Egyptian children and adults by an oral,    killed enterotoxigenic Escherichia coli plus cholera toxin B subunit    vaccine, Infect Immun, 69:2853-2857.-   Hammond et al. (2000) Transcutaneous immunization of domestic    animals: opportunities and challenges, Adv Drug Delivery Rev,    43:45-55.-   Hammond et al. (2001) Transcutaneous immunization: T cell responses    and boosting of existing immunity, Vaccine, 19:2701-2707.-   Harlow & Lane (1988) Antibodies: A laboratory manual. Cold Spring    Harbor Press.-   Hartman et al. (1994) Local immune response and protection in the    guinea pig keratoconjunctivitis model following immunization with    Shigella vaccines, Infect Immun, 62:412-420.-   Hartman et al. (1999) Native and mutant forms of cholera toxin and    heat-labile enterotoxin effectively enhance protective efficacy of    live attenuated and heat-killed Shigella vaccines, Infect Immun,    67:5841-5847.-   Helander et al. (1997) Binding of enterotoxigenic Escherichia coli    to isolated enterocytes and intestinal mucus, Microb Pathogen,    23:335-346.-   Helander et al. (1998) Antibody responses in humans against coli    surface antigen 6 of enterotoxigenic Escherichia coli, Infect Immun,    66:4507-4510.-   Hoffman et al. (1994) Safety, immunogenicity, and efficacy of a    malaria sporozoite vaccine administered with monophosphoryl lipid A,    cell wall skeleton of mycobacteria, and squalane as adjuvant, Am J    Trop Med Hyg, 51:603-612.-   Hoge et al. (1998) Trends in antibiotic resistance among diarrheal    pathogens isolated in Thailand over 15 years, Clin Infect Dis,    26:341-345.-   Huerta et al. (2000) A waterborne outbreak of gastroenteritis in the    Golan Heights due to enterotoxigenic Escherichia coli, Infect,    28:267-271-   Hyams et al. (1991) Diarrheal disease during Operation Desert    Shield, N Engl J Med, 325:1423-1428.-   Jacobs et al. (1982) Adverse reactions to tetanus toxoid, JAMA,    247:40-42.-   Jertborn et al. (1986) Saliva, breast milk, and serum antibody    responses as indirect measures of intestinal immunity after oral    cholera vaccination or natural disease, J Clin Microbiol,    24:203-209.-   Jertborn et al. (1998) Safety and immunogenicity of an oral    inactivated enterotoxigenic Escherichia coli vaccine, Vaccine,    16:255-260.-   Jertborn et al. (2001) Dose-dependent circulating immunoglobulin A    antibody-secreting cell and serum antibody responses in Swedish    volunteers to an oral inactivated enterotoxigenic Escherichia coli    vaccine, Clin Diag Lab Immunol, 8:424-428.-   Jiang et al. (2000) Characterization of enterotoxigenic Escherichia    coli strains in patients with travelers' diarrhea acquired in    Guadalajara, Mexico, 1992-1997, J Infect Dis, 181:779-782.-   Jodar et al. (2001) Ensuring vaccine safety in immunization    programmes—a WHO perspective, Vaccine, 19:1594-1605.-   Keitel et al. (1993.) Pilot evaluation of influenza virus vaccine    (IVV) combined with adjuvant, Vaccine, 11:909-913.-   Levine (1981) Adhesion of enterotoxigenic Escherichia coli in humans    and animals, Ciba Found Symp, 80:142-60.-   Levine (1983) Travellers' diarrhoea: prospects for successful    immunoprophylaxis, Scand J Gastroenterol—Suppl, 84:121-134.-   Levine et al. (1979) Immunity to enterotoxigenic Escherichia coli,    Infect Immun, 23:729-736.-   Levine et al. (1983) New knowledge on pathogenesis of bacterial    enteric infections as applied to vaccine development, Microbiol Rev,    47:510-550.-   Lycke (1997) The mechanism of cholera toxin adjuvanticity, Res    Immunol, 148:504-520.-   Mattila et al. (1992) Seasonal variation in etiology of travelers'    diarrhea. Finnish-Moroccan Study Group, J Infect Dis, 165:385-388.-   Michetti et al. (1999) Oral immunization with urease and Escherichia    coli heat-labile enterotoxin is safe and immunogenic in Helicobacter    pylori-infected adults, Gastroenterol, 116:804-812.-   Murphy et al. (2001) Treatment of traveler's diarrhea, pp. 165-176.    In H. L. DuPont, Stephen, R. (ed.), Texbook of travel medicine and    health. Books News Inc., Portland.-   Nagy & Fekete (1999) Enterotoxigenic Escherichia coli (ETEC) in farm    animals. Vet. Res. 30:259-284.-   Orndorff et al. (1996) Enterotoxigenic Escherichia coli diarrhea in    children less than five years of age in central Java, Am J Trop Med    Hyg, 55:449-451.-   Pierce & Reynolds (1974) Immunity to experimental cholera. I.    Protective effect of humoral IgG antitoxin demonstrated by passive    immunization, J Immunol, 113:1017-1023.-   Pierce & Reynolds (1974) Immunity to experimental cholera. I.    Protective effect of humoral IgG antitoxin demonstrated by passive    immunization, J Immunol, 113:1017-1023.-   Pierce et al. (1972) Protection against experimental cholera by    antitoxin, J Infect Dis, 126:606-616.-   Pierce et al. (1980) Antitoxic immunity to cholera in dogs immunized    orally with cholera toxin, Infect Immun, 27:632-637.-   Qadri et al. (2000) Safety and immunogenicity of an oral,    inactivated enterotoxigenic Escherichia coli plus cholera toxin B    subunit vaccine in Bangladeshi adults and children, Vaccine,    18:2704-2712.-   Richardson et al. (1984) Sealed adult mice: new model for    enterotoxin evaluation, Infect Immun, 43:482-486.-   Sack et al. (1984) Doxycycline prophylaxis of travelers' diarrhea in    Honduras, an area where resistance to doxycycline is common among    enterotoxigenic Escherichia coli, Am J Trop Med Hyg, 33:460-466.-   Sahai & Khurshid (1995) On analysis of epidemiological data    involving a 2×2 contingency table: an overview of Fisher's exact    test and Yates' correction for continuity, J Biopharm Stat, 5:43-70.-   Savarino et al. (1998) Safety and immunogenicity of an oral, killed    enterotoxigenic Escherichia coli-cholera toxin B subunit vaccine in    Egyptian adults, J Infect Dis, 177:796-799.-   Savarino et al. (1999) Oral, inactivated, whole cell enterotoxigenic    Escherichia coli plus cholera toxin B subunit vaccine: results of    the initial evaluation in children. PRIDE Study Group, J Infect Dis,    179:107-114.-   Schagger & von Jagow (1987) Tricine-sodium dodecyl    sulfate-polyacrylamide gel electrophoresis for the separation of    proteins in the range from 1 to 100 kDa, Anal Biochem, 166:368-379.-   Scharton-Kersten et al. (2000) Transcutaneous immunization with    bacterial ADP-ribosylating exotoxins, subunits, and unrelated    adjuvants, Infect Immun, 68:5306-5313.-   Schultsz et al. (2000) Diarrheagenic Escherichia coli and acute and    persistent diarrhea in returned travelers, J Clin Microbiol,    38:3550-3554.-   Schultz et al. (1995) Effect of DETOX as an adjuvant for melanoma    vaccine, Vaccine, 13:503-508.-   Scott et al. (1990) Norfloxacin for the prophylaxis of travelers'    diarrhea in U.S. military personnel, Am J Trop Med Hyg, 42:160-164.-   Stoll et al. (1986) Local and systemic antibody responses to    naturally acquired enterotoxigenic Escherichia coli diarrhea in an    endemic area, J Infect Dis, 153:527-534.-   Svennerholm et al. (1983) Serologic differentiation between    antitoxin responses to infection with Vibrio cholerae and    enterotoxin-producing Escherichia coli, J Infect Dis, 147:514-522.-   Tacket et al. (1988) Protection by milk immunoglobulin concentrate    against oral challenge with enterotoxigenic Escherichia coli, New    Engl J Med, 318:1240-1243.-   Tacket et al. (1994) Enteral immunization and challenge of    volunteers given enterotoxigenic E. coli CFA/II encapsulated in    biodegradable microspheres, Vaccine, 12:1270-1274.-   Tacket et al. (1999) Lack of prophylactic efficacy of an    enteric-coated bovine hyperimmune milk product against    enterotoxigenic Escherichia coli challenge administered during a    standard meal, J Infect Dis, 180:2056-2059.-   Taylor et al. (1985) Polymicrobial aetiology of travellers'    diarrhoea, Lancet. 1:381-383.-   Todd (1997) Epidemiology of foodborne diseases: A worldwide review,    World Health Statistics Quarterly, 50:30-50.-   Trach et al. (1997) Field trial of a locally produced, killed, oral    cholera vaccine in Vietnam, Lancet, 349:231-235.-   Vassell et al. (1999) Activation of Langerhans cells following    transcutaneous immunization, pg. 13. The 5th National Symprosium,    Basic Aspects of Vaccines, Bethesda, Md.-   Wenneras et al. (1992) Antibody-secreting cells in human peripheral    blood after oral immunization with an inactivated enterotoxigenic    Escherichia coli vaccine, Infect Immun, 60:2605-2611.-   Wolf (1997) Occurrence, distribution, and associations of O and H    serogroups, colonization factor antigens, and toxins of    enterotoxigenic Escherichia coli. Clin. Microbiol Rev, 10:569-584.-   Wolf (1997) Occurrence, distribution, and associations of O and H    serogroups, colonization factor antigens, and toxins of    enterotoxigenic Escherichia coli, Clin Microbiol Rev, 10:569-584.-   Wolf et al. (1993) Characterization of enterotoxigenic Escherichia    coli isolated from U.S. troops deployed to the Middle East. J Clin    Microbiol, 31:851-856.-   Wolf et al. (1997) The CS6 colonization factor of human    enterotoxigenic Escherichia coli contains two heterologous major    subunits. FEMS Microbiol Lett, 148:35-42.-   Wolf et al. (1997) U.S. Pat. No. 5,698,416. Methods for production    of antigens under control of temperature-regulated promoters in    enteric bacteria. Walter Reed Army Institute of Research.-   Wolf et al. (1999) Use of the human challenge model to characterize    the immune response to the colonization factors of enterotoxigenic    Escherichia coli (ETEC) The 35th Joint Conference of the U.S.-Japan    Cooperative Medical Science Program, Tokyo, Japan.-   Wood et al. (1983) Antimicrobial resistance of gram-negative    bacteria isolated from foods in Mexico, J Infect Dis, 148:766.-   Yu, J., F. J. Cassels, T. Scharton-Kersten, S. A. Hammond, A.    Hartman, E. Angov, C. Corthesy, C. R. Alving, and G. M. Glenn    Transcutaneous immunization using colonization factor and heat    labile enterotoxin induces correlates of protective immunity for    enterotoxigenic Escherichia coli. Infect Immun, in press.

All references (e.g., articles, books, patents, and patent applications)cited above are indicative of the level of skill in the art and areincorporated by reference.

All modifications and substitutions that come within the meaning of theclaims and the range of their legal equivalents are to be embracedwithin their scope. A claim using the transition “comprising” allows theinclusion of other elements to be within the scope of the claim; theinvention is also described by such claims using the transitional phrase“consisting essentially of” (i.e., allowing the inclusion of otherelements to be within the scope of the claim if they do not materiallyaffect operation of the invention) and the transition “consisting”(i.e., allowing only the elements listed in the claim other thanimpurities or inconsequential activities which are ordinarily associatedwith the invention) instead of the “comprising” term. No particularrelationship between or among limitations of a claim is meant unlesssuch relationship is explicitly recited in the claim (e.g., thearrangement of components in a product claim or order of steps in amethod claim is not a limitation of the claim unless explicitly statedto be so). Thus, all possible combinations and permutations of theindividual elements disclosed herein are intended to be considered partof the invention.

From the foregoing, it would be apparent to a person of skill in thisart that the invention can be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments should be considered only as illustrative, not restrictive,because the scope of the legal protection provided for the inventionwill be indicated by the appended claims rather than by thisspecification

1. A method of preventing traveler's diarrhea in a human that is to besubjected to exposure to pathogens causing traveler's diarrheacomprising applying a vaccine transcutaneously to the skin of the human,prior to exposure to pathogens causing traveler's diarrhea, wherein thevaccine comprises an effective amount of heat-labile enterotoxin of E.coli (LT) to prevent traveler's diarrhea.
 2. A method of preventingtraveler's diarrhea in a human that is to be subjected to exposure topathogens causing traveler's diarrhea comprising applying a vaccinetranscutaneously to the skin of the human, prior to exposure topathogens causing traveler's diarrhea, wherein the vaccine comprises aneffective amount of heat-labile enterotoxin of E. coli (LT) to treattraveler's diarrhea.
 3. The method of claim 1 or 2, wherein the vaccinecomprises LT and an adjuvant.
 4. The method of claim 3, wherein theadjuvant is an ADP-ribosylating exotoxin or a derivative thereof havingadjuvant activity.
 5. The method of claim 1 or 2, wherein the vaccinecomprises LT and an E. coli colonization factor antigen (CFA).
 6. Themethod of claim 5, wherein the E. coli colonization factor antigen isselected from the group consisting of CFA/I, CS1, CS2, CS 4, CS5, CS6,CS17 and PCF
 0166. 7. The method of claim 1, wherein the vaccinecomprises LT and heat stable enterotoxin of E. coli (ST).
 8. The methodof claim 7, wherein the vaccine comprises a carrier or an excipient. 9.The method of claim 7, wherein LT is conjugated to ST.
 10. The method ofclaim 1, wherein the method comprises pretreating the skin prior toadministering the vaccine.
 11. The method of claim 10, whereinpretreating comprises chemical penetration enhancement, physicalpenetration enhancement, or both.
 12. The method of claim 1 or 2,wherein the vaccine is applied using a patch.
 13. The method of claim 12wherein the vaccine comprises a carrier or an excipient.
 14. The methodof claim 1, wherein LT is genetically detoxified.
 15. The method ofclaim 1 or 2, wherein LT is a mutant form of the enterotoxin.
 16. Themethod of claim 1 or 2, wherein LT is both an antigen and an adjuvant.17. The method of claim 16, wherein LT is a mutant form of theenterotoxin.